Rationale: Chronic wounds are one of the common complications of diabetes. Due to the physiological conditions of diabetic patients, these wounds are more susceptible to bacterial infections and the formation of bacterial biofilms, leading to the inefficiency of conventional antibiotic treatment.Methods: Here, hollow mesoporous silica nanoparticles (HMSN) were used as the nanocarriers for co-delivery of azithromycin (AZM) and glucose oxidase (GOX), achieving a remarkable synergistic effect in chronic diabetic wounds. GOX possesses the catalytic ability to consume glucose and produce H2O2 in the diabetic wound area. The down-regulation of local glucose could effectively improve the chronic diabetic wound microenvironment. Meanwhile, the generated H2O2 effectively inhibits bacterial growth and eradicates bacterial biofilms with the synergism of antibiotics AZM.Results: In the bacteria-infected diabetic cutaneous wound models, the reduction of glucose, generation of H2O2, and release of AZM could effectively reduce the bacterial infection and promote the wounds healing. Moreover, there is no obvious toxicity behavior after the treatment.Conclusions: Therefore, the designed nanosystem could effectively accelerate the diabetic wound healing process by the amelioration of the hyperglycemia microenvironment and the eradication of bacterial biofilms around the wounds, making them promising candidates for clinical transformation. Chronic wounds are one of the major complications associated with diabetes, which represent a huge burden for both affected individuals and the entire healthcare system 2O2) with the assistance of oxygen 2O2. It is reported that H2O2 along with its secondary metabolite hydroxyl radical could damage bacteria cell components, leading to the breakdown of bacteria cell membranes and walls 2O2 promotes wound healing by mediating leukocyte recruitment, facilitates the release of vascular endothelial growth factor and improved blood flow 2O2. Therefore, GOX is expected to ameliorate the conditions of diabetic wounds and enhance the therapeutic efficacy of antibiotics.Glucose oxidase (GOX) is an endogenous oxidoreductase that can specially catalyze glucose oxidation into gluconic acid and hydrogen peroxide  that possess antibacterial effects and local glucose reduction simultaneously to facilitate bacteria-infected diabetic wound healing. Due to the special spatial structure, high specific surface area and modification sites of HMSN, it enables both the effective loading of AZM into its internal cavity and the GOX functionalization of the material surface. On the one hand, GOX can react with glucose to produce HS. aureus, ATCC 6538) was obtained from the American type culture collection . Phosphate-buffered saline  was purchased from Cienry . Glucose, Luria-Bertani (LB) broth and LB broth agar were purchased from Sangon Biotech . Live/Dead BacLight bacterial viability kit was purchased from Thermo Fisher Scientific. Db/db mice were obtained from SLAC Laboratory Animal Co., Ltd . Purified water was used throughout.Glucose oxidase (GOX) and (3-Aminopropyl) triethoxysilane  were purchased from Sigma. Azithromycin  and triethanolamine  were purchased from Macklin . Tetraethyl orthosilicate  and hexadecyltrimethylammonium chloride  were purchased from Aladdin. Staphylococcus aureus (2 (dSiO2) nanoparticles were prepared according to a modified Stöber method. The mixed solution of 142.8 mL ethanol, 20 mL water and 3.2 mL ammonia were stirred for 10 minutes at room temperature, followed by the addition of 4 mL TEOS. Then the mixture was stirred for 1h. dSiO2 nanoparticles were collected by centrifugation, washed with water twice and suspended in 40 mL water. Secondly, the synthesis of dSiO2@MSN. 16 g CTAC and 160 mg TEA were mixed in 200 mL water and stirred at 50 ºC. After mixing completely, 40 mL dSiO2 solution was added and stirred for 1 h at 80 ºC. Then 1.2 mL TEOS was dropwise added and kept stirring for 1 h. Thirdly, HMSN was obtained by etching dSiO2@MSN 2CO3 was used to selectively etch the internal dSiO2 core and the mixture was stirred for 30 min. The resulting HMSN were collected by centrifugation  and then were extracted with a methanol solution of NaCl (1 wt %) for 24 h at room temperature three times to remove CTAC. Finally, the HMSN were dispersed in the water.Firstly, dense SiOTo load AZM into the cavity of HMSN, 50 mg HMSN and the appropriate amount of AZM were dissolved in ethanol and stirred for 24 h at room temperature. The resulting HMSN-AZM were collected by centrifugation  and washed with water. The standard solution of AZM was prepared for UV-vis determination to construct a standard curve. AZM loading capacity was obtained by measuring the AZM concentration of the supernatant before and after loading.GOX was functionalized onto the surface of HMSN by covalent grafting according to the literature method 95 mg EDC, 142.5 mg NHS and 10 mg GOX were dissolved in 15 mL water, followed by the addition of 112 µL APTES. The mixture was stirred for 8 h at room temperature to produce amino-functionalized GOX. Then 50 mg HMSN was added to the mixture and stirred for 24 h. The final nanoparticles (GOX-HMSN) were collected by centrifugation  and washed with water three times.The morphology of the nanoparticle and bacteria were characterized by Transmission electron microscopy . Hydrodynamic size and zeta potential measurements were performed at room temperature by a dynamic light scattering system . XRD patterns were characterized by the X-ray diffractometer . Raman spectra were obtained by Raman spectrometer . UV-Vis spectra were recorded with an ultraviolet spectrophotometer . FTIR patterns were characterized on an FTIR Spectrometer .20 mM glucose solution was prepared, which was co-cultured with different concentrations  of GOX-HMSN or GOX-HMSN-AZM at 37 ºC for 12 h. Then, a glucometer  was used to measure the glucose concentration of the different groups. Besides, 20 mM glucose solution was cultured with 200 μg/mL of GOX-HMSN or GOX-HMSN-AZM at 37 ºC. The glucose concentration at different incubating times  were recorded using a glucometer (n = 3).4)2 and 8.33 mL of H2SO4 were added in 50 mL distilled water to prepare the Ti(SO4)2 cocktail detection solution. The standard solutions of glucose were mixed with Ti(SO4)2 cocktail detection solutions (1:1), and the corresponding UV-vis absorptions at 405 nm were recorded to construct an H2O2 standard curve. Then, 20 mM glucose solution was prepared, which was cultured with different concentrations  of GOX-HMSN or GOX-HMSN-AZM at 37 ºC for different times. The supernatant was collected by centrifugation  and then was mixed with Ti(SO4)2 cocktail detection solution (1:1). The absorbance was measured at 405 nm and then the concentration of H2O2 was acquired by the standard curve (n = 3).Firstly, 1.33 mL of 24% Ti of GOX-HMSN or GOX-HMSN-AZM at 37 ºC for 12 h. Afterwards, a pH meter  was used to measure the pH values. Besides, 20 mM glucose solution was cultured with 200 μg/mL of GOX-HMSN or GOX-HMSN-AZM at 37 ºC. Using the pH meter to measure the pH variation at different incubating times  (n = 3).The standard solution of AZM was prepared and the corresponding UV-vis absorptions at 215 nm were recorded to construct a standard curve. The HMSN-AZM and GOX-HMSN-AZM were incubated with glucose (20 mM) at 37 ºC. At predetermined time points , the supernatant of the solution was collected by centrifugation  and then evaporated. Ethanol was used to dissolve the released AZM. The absorbance was measured and then the concentration of AZM was acquired by the standard curve (n = 3).The concentration of GOX was determined using a BCA protein assay kit. Protein standards (0、1、2、4、8、12、16、20 μL), GOX-HMSN (20 μL) and GOX-HMSN-AZM (20 μL) were added into 96-wells plates (n=3). Then 200 μL working reagent was added to each well and mixed. The solution was incubated at 60 ºC for 30 min. A multimode microplate reader was used to measure the optical density at 562 nm. Then the standard curve of protein was prepared and the GOX concentration was determined by the standard curve (n = 3).4)2 cocktail detection solution was added to measure the production of H2O2. The absorbance was measured at 405 nm and then the concentration of H2O2 was acquired by the standard curve. The data were analyzed using the Lineweaver-Burk plot and the kinetic parameters Km and Kcat were calculated using the Michaelis-Menten Equation (n = 3).For the enzyme kinetic measurements of the nanocomposites, 0-30 mM glucose was used as substrate. GOX-HMSN and GOX-HMSN-AZM were incubated with different concentrations of glucose at 37 ºC for 30 min. The supernatant was collected by centrifugation . Then the Ti(SO4)2 cocktail detection solution (1:1). The absorbance was measured at 405 nm and the concentration of H2O2 was acquired by the standard curve (n = 3).GOX-HMSN and GOX-HMSN-AZM nanoparticles were stored at 4 ºC. After 0, 3, 7, 14 and 30 days, 20 mM glucose solution was mixed with the nanoparticles to initiate the reaction. The supernatant was collected by centrifugation  and then was mixed with Ti(SOS. aureus suspension (109 CFU/mL) was cultured with different concentrations  of HMSN, HMSN-AZM, GOX-HMSN and GOX-HMSN-AZM in LB broth (20 mM Glucose contained) for 24 h at 37 ºC. After incubation, a multimode microplate reader  was used to measure the bacteria concentration by determining the optical density at 600 nm (n = 4).S. aureus suspension  was cultured with different concentrations of AZM and GOX-HMSN-AZM in LB broth (20 mM Glucose contained) for 24 h at 37 ºC. Then the bacteria survived after half MIC (passage 1) treatment were designated as passage 2. The above operations were repeated for 10 successive passages (n=3).A minimum inhibitory concentration (MIC) test was used to evaluate the bacterial resistance. Firstly, S. aureus suspension (109 CFU/mL) was incubated with HMSN, HMSN-AZM, GOX-MSN and GOX-HMSN-AZM at 15.6 μg/mL for 3 h at 37 ºC. Then SYTO 9/PI live/dead bacteria viability kit was used following the corresponding instruction. Briefly, SYTO 9 and PI were diluted with PBS (1:1000) and then mixed with the treated bacterial suspension above for 20 min at room temperature. After that, the bacterial suspension was placed on glass slides. Images were obtained with a fluorescence confocal microscope .S. aureus suspension was diluted to 109 CFU/mL, then was cultured with 15.6 μg/mL HMSN, HMSN-AZM, GOX-HMSN, GOX-HMSN-AZM for 6 h. The suspension was collected by centrifugation and fixed by glutaraldehyde solution (2.5%) for 12 h. Afterwards, the sample was washed three times with PBS for 15 min and embossed with agar. Then the sample was fixed by osmic acid for 2 h, followed by a gradient of dehydration with an ethanol-water mixture  for 10 min in turn. Finally, the dried sample was sputter-coated with gold and analyzed by TEM .S. aureus suspension (107 CFU/mL) was incubated in 96-wells plates with LB broth (20 mM glucose contained) for 24 h to form bacterial biofilms. Then the medium was replaced by fresh LB broth (20 mM glucose contained) with different concentrations  of HMSN, HMSN-AZM, GOX-HMSN and GOX-HMSN-AZM. After 24 h of incubation, the wells were washed by PBS for twice to remove free bacteria. Subsequently, the bacterial biofilms were stained with 100 μL crystal violet (1%) solution for 20 min at room temperature and then washed with PBS three times to remove excess dye. Then, 100 μL ethanol was added to each well and incubated for 10 min at room temperature. The optical densities of crystal violet were measured by the multimode microplate reader at 570 nm. In addition, the standard dilution plate assay was used to evaluate the effects against bacterial biofilms quantitatively. The bacterial biofilms were transferred into the centrifuge tube. After 10 min of ultrasonication, the living bacteria were obtained by gradient dilution and plate counting method (n = 3).Biofilm formation was detected by the crystal violet method. Briefly, S. aureus suspension (107 CFU/mL) was incubated in 12-wells plates with LB broth (20 mM glucose contained) for 24 h to form bacterial biofilms. Then the medium was replaced by fresh LB broth (20 mM glucose contained) with 500 μg/mL HMSN, HMSN-AZM, GOX-HMSN and GOX-HMSN-AZM. After 24 h of incubation, the wells were washed by PBS for twice to remove free bacteria. Then SYTO 9 bacterial viability kit was used to stain bacterial biofilms. Briefly, SYTO 9 was diluted with PBS (1:1000) and then added 1 mL of SYTO 9 solution to each well for 20 min at room temperature. The biofilm morphology was examined under a 3D confocal scanning microscopy  later.S. aureus suspension . After 24 h, mice were treated with HMSN, HMSN-AZM, GOX-HMSN and GOX-HMSN-AZM  respectively except that the control group was treated with PBS. Wound areas were photographed and measured at different times after treatments. Then the wound tissues were excised for pathological histology and immunohistochemistry analysis after 14 days of treatments. These tissues were washed with PBS and fixed in 10% formalin. Then the treated tissues were embedded in paraffin and sectioned into 5 μm sections. These sections were analyzed with hematoxylin and eosin (H&E), Masson's trichrome staining and immunohistochemistry method . All sections were examined by a virtual digital slide scanning system.All animal experiments were approved by the institutional animal care and use committee of Zhejiang University  and were individually raised in cages under standardized temperature. Db/db mice  were anesthetized by inhalation of isoflurane and shaved on the dorsum. Round skin wound injury (diameter of 10 mm) was generated with a surgical scalpel. Then the mice (n=3) were divided into different groups randomly. Skin wounds of all groups were infected by After the above treatment of wounds (14 days), the blood samples were obtained for clinical chemistry and hematologic analysis. Then major organs  were harvested from mice in each group, washed with PBS and fixed in 10% formalin. Then the treated tissues were embedded in paraffin and sectioned into 5 μm sections. These sections were stained with H&E for toxicology histological analysis. All sections were examined by a virtual digital slide scanning system.Scheme . Firstly, uniform dense SiO2 (dSiO2) nanoparticles about 112 nm in size were synthesized as hard templates, which possessed negative surface charge (-47.0 mV) . Secondly, the in situ coating of a mesoporous silica shell on and around dSiO2 nanoparticles was carried out . Finally, dSiO2 and soft templates (CTAC) were selectively removed to generate the HMSN nanoparticles.It is well known that silicon nanoparticles have been widely employed as drug delivery platforms due to their excellent loading capacity, biocompatibility, and biodegradability Figure  showed that the as-synthesized HMSN has a huge cavity, which makes it promise candidate for drug loading. Brunner-Emmet-Teller (BET) measurements show that HMSN has a relatively large surface area of 617 m2/g and a pore size of 3.76 nm , which facilitates efficient loading of guest molecules into the hollow cavity. Here, AZM was selected to be loaded into the HMSN by a soaking method, yielding HMSN-AZM. The loading capacity of AZM calculated from the UV-vis absorption standard curve was proved to be about 12.5 w/w% . Then, GOX was modified on the surface of HMSN-AZM to obtain GOX-HMSN-AZM, endowing the nanoparticles with the catalytic ability. As shown in Figure  AZM loading and GOX modification do not affect the structure and morphology of the nanoparticles. The high-angle annular dark-field (HAADF) STEM image of GOX-HMSN-AZM in Figure  also shows a clear hollow nanostructure, where the distribution of three major elements  is uniform . The hydrodynamic diameter of the nanoparticles also shows negligible changes after loading and modification, indicating the high dispersity of the nanoparticles . As shown in Figure , the zeta potential of nanoparticles transformed from -23.3 mV to 27.8 mV after loading and modification, which demonstrated the successful preparation of GOX-HMSN-AZM. The X-ray diffraction (XRD) patterns  further confirm that AZM had been successfully encapsulated into the HMSN. Raman spectroscopic investigation was also conducted. The characteristic peaks of AZM appear at 1450 cm-1 and 2934 cm-1 , which demonstrates the successful loading of AZM. UV-vis spectra confirmed that GOX has been successfully combined on the surface of HMSN-AZM . FTIR analysis was further carried out to verify the modification of GOX . The new peak at 1537 cm-1 is corresponding to the characteristic peak of GOX . Altogether, the results above indicate that we have successfully designed and prepared monodisperse GOX-HMSN-AZM nanoparticles.Transmission electron microscope (TEM) images in 2O2, the catalytic capacity of GOX-HMSN-AZM was evaluated by measuring the glucose consumption, gluconic acid production and H2O2 production. Firstly, the consumption of glucose was evaluated during the reaction by a glucometer. With the accumulation of time, glucose which was incubated with GOX-HMSN-AZM at 200 μg/mL gradually reduced . At the same reaction time for 12 h, glucose concentration reduced along with the increasing concentrations of the GOX-HMSN-AZM nanoparticles . Then, the gluconic acid production was assessed by the detection of pH variation. The solution changed from neutral (~7.26) to acidic (~3.15) within 24 hours . The pH value declined quickly in the first three hours and then changed tardily. At a fixed reaction time for 12 h, the pH of the solution decreased along with the GOX-HMSN-AZM concentration increased . Subsequently, the titanium sulfate colorimetric method 2O2 . Under the catalyst of 200 μg/mL GOX-HMSN-AZM, H2O2 is produced sustainably over time . As shown in Figure  and , the generated H2O2 was increased in response to the elevated concentrations of GOX-HMSN-AZM which was incubated with glucose for 12 hours.Since GOX can specially catalyze glucose oxidation into gluconic acid and HTable S1). Km value represents the binding affinity of the enzyme to the substrate and Kcat/Km value is defined as the catalytic efficiency of enzymatic reaction. Therefore, owing to the spatial separation, the loading of AZM did not interfere with the activity of GOX, leading to the equivalent catalytic efficiency between GOX-HMSN and GOX-HMSN-AZM. The storage stability of both GOX-HMSN and GOX-HMSN-AZM was also evaluated. After 30-day storage at 4 ºC, GOX-HMSN and GOX-HMSN-AZM retained 73.9% and 70.4% of initial enzymatic activity respectively .In order to further understand the reactivity of the encapsulated enzymes, we evaluated the kinetic parameters utilizing the GOX-HMSN and GOX-HMSN-AZM  infection is reported as the commonest bacterial infection in diabetic wounds  S. aureus. As shown in Figure  the bacterial survival rate of HMSN treaded group showed no obvious difference with the untreated control group, indicating the non-toxicity of HMSN as the nanocarrier. Due to the low water solubility, free AZM demonstrated poor antibacterial efficacy . It is worth noting that, with the nanoparticle concentration increases, HMAN-AZM showed a limited antimicrobial effect in high glucose environment. Meanwhile, GOX-HMSN showed improved antimicrobial activity, but it was still limited at low concentrations. However, GOX-HMSN-AZM showed significant improvement in antibacterial capability and could significantly reduce the number of viable bacteria at a low concentration of 15.63 μg/mL . Furthermore, the bacterial resistance test was occupied. As shown in Figure S11, the MIC value of S. aureus incubated with free AZM increased 80-fold after 10 passages incubation, indicating serious drug resistance. Notably, the MIC value of S. aureus incubated with free GOX-HMSN-AZM increased 16-fold compared to passage 1.Due to the declined ability to metabolize glucose, the wound conditions of diabetic patients tend to be complicated S. aureus with different treatments. As shown in Figure , the fluorescence intensities of SYTO9 (green) and PI (red) in all the five groups were consistent with the bacterial survival assay results. Scarcely any bacteria stained by PI (red fluorescence) could be observed in control, HMSN treated groups. Compare with the HMSN-AZM and GOX-HMSN groups, an increased red fluorescence could be observed in GOX-HMSN-AZM group, which demonstrated the rapid antibacterial efficiency of GOX-HMSN-AZM nanoparticles in the hyperglycemia environment.Then, the SYTO9/PI live/dead fluorescent staining assay was employed in the current study to analyze the survival of Figure , the cytoplasm of the bacteria incubated with GOX-HMSN and GOX-HMSN-AZM partially formed aggregations, indicating the damage of the cells. It is worth noting that the bacterial membranes were most lysed after GOX-HMSN-AZM treatments, leading to the loss of structural integrity of bacterial cell walls. Taken together, these results suggested the strong interaction between GOX-HMSN-AZM and bacteria by disrupting the bacteria cell wall and membrane integrity, indicating the remarkably improved synergistic therapeutic effects of antibiotics and enzymatic reactions, much better than either single treatment alone.To further study the antibacterial process of the GOX-HMSN-AZM nanoparticles, the structure variation of the bacteria was employed by TEM. As shown in Traditional antibiotic treatment is often less effective in diabetics owing to the presence of bacterial biofilms in the wound S. aureus biofilms was evaluated by crystal violet staining assay. As shown in Figure , after treatment with the HMSN and HMSN-AZM, the biofilm structure exhibited negligible changes at tested doses. However, significant biofilm destructions were generated with the treatments of GOX-HMSN and GOX-HMSN-AZM at high concentrations (500 μg/mL). It is worth noting that GOX-HMSN-AZM exhibit a much stronger biofilm destruction capability than the GOX-HMSN even at the same concentrations. The corresponding statistic results in Figure  and Figure S12 indicate the percentage of biofilm remaining after treatments. There was no significant difference in the biofilm mass residuals treated with HMSN and HMSN-AZM compared to the control. At the concentrations of 500 and 1000 μg/mL, GOX-HMSN-AZM destroyed around 85.3% and 92.1% of the biofilms, which was significantly higher than that with the treatment of GOX-HMSN.The disruption effect of different treatments against S. aureus biofilm. As shown in Figure , the quantified results showed that when bacteria were treated with HMSN, there is no significant difference between the number of colonies compared to that of the control. Due to the inherent high antibiotic resistance of biofilms, only 28.6% of bacteria were killed after biofilms were treated with HMSN-AZM at 500 μg/mL. GOX-HMSN showed relatively efficient antibacterial ability, which eliminated 93.3% of S. aureus in biofilms. Notably, GOX-HMSN-AZM exhibited better antibacterial ability against S. aureus biofilms, which caused a 99.4% reduction of bacterial survival rate at the same concentration.Then the dilution plate assay was employed to further quantify the disruption of different treatments against S. aureus biofilm after different treatments through intuitively observing the variation of biofilm thickness and density. As shown in Figure -G, the structure of the bacterial biofilms treated with HMSN and HMSN-AZM was intact and compact, which was similar to the control. The treatment of GOX-HMSN eradicated the bacterial biofilm to some extent. In comparison, the bacterial biofilms treated with GOX-HMSN-AZM were thinnest and sparsest among all. Quantitative analysis demonstrated that the thickness of the bacterial biofilms treated with GOX-HMSN-AZM reduced 75.2% and the relative fluorescence intensity reduced 79.8% in comparison to the control group , which demonstrated the efficient eradication induced by our strategy. The results above demonstrated that GOX-HMSN-AZM effectively eradicated bacterial biofilm, which was beneficial for treating bacterial infections in chronic diabetic wounds.Ultimately, confocal microscopy was applied to further verify the destruction of in vitro results, we further verified whether the composite nanoparticles can accelerate the wound healing process in diabetic infectious wounds. Thus, the bacteria-infected cutaneous wound model in diabetes (db/db) mice was used to evaluate the antibacterial effects and wound healing effects of our strategy. The treatments were applied 24 hours after infection, when the bacterial biofilms were formed in the wound areas . Representative pictures of the infected wounds were shown in Figure  and the wound areas were also measured . The wounds of mice treated with GOX-HMSN-AZM almost removed the infection and healed the wounds after 14 days, whereas other treatments induced incomplete recovery. The HMSN-AZM and GOX-HMSN groups had a therapeutic effect on diabetic wounds to some extent. However, due to the limited therapeutic effect of individual treatments, it could be observed the invasive and persistent bacterial infection in the surrounding area around the wound in the diabetic mice in the group of HMSN-AZM, and GOX-HMSN. It is noteworthy that the diabetic wounds treated with GOX-HMSN-AZM exhibited speedy and efficacious recovery due to the high antibacterial and antibiofilm activity of GOX-HMSN-AZM in hyperglycemia environment.Due to the complicated physiological conditions related to hyperglycemia, diabetic wounds generate prolonged inflammation, recurrent infections which greatly extend the treatment period Figure ). For the untreated group, the histological slice displayed the obvious infiltration of inflammatory cells, indicating an intense immune response against bacterial infection. In contrast, fewer inflammatory cells were observed in the group treated with GOX-HMSN-AZM, which demonstrated the effective antimicrobial and infection suppression of the treatment strategy. Moreover, the collagen fibers in the wound tissue treated with GOX-HMSN-AZM were dense and ordered, indicating superior wound repair in comparison to other treatment groups .Subsequently, the wound epithelial tissue was excised and stained for histological analysis. Hematoxylin and eosin-stained section (H&E) demonstrated the inflammation status of the wound  was assessed using immunohistochemical staining of CD31 Figure  and Figure S14, FGF was also significantly upregulated in GOX-HMSN-AZM group. Taken together, these results demonstrated that GOX-HMSN-AZM could accelerate the wound healing process of bacteria-infected diabetic mice by reducing the burden of bacterial infection and promoting the formation of more vessels.To evaluate the mechanism of GOX-HMSN-AZM in promoting wound healing, tissue slices collected from different groups were analyzed by immunohistochemistry. It is generally acknowledged that the process of angiogenesis is vital for optimal wound repair Figure S15). Figure  showed no noticeable organ damage or inflammatory lesions in histology analysis between each group of mice. Besides, there were no significant changes in blood biochemistry and hematology examination . These results above demonstrated the safety and high biocompatibility of GOX-HMSN-AZM in vivo, showing the great potential for our strategy in future clinical translation.The safety of the treatments was further assessed. During the entire therapy period, the body weight of the treated mice was stable and there were no significant differences between the groups (2O2, simultaneously. The increased acidity by the catalytic reaction also promotes the release of AZM. In the bacteria-infected diabetic cutaneous wound models, the reduction of glucose, generation of H2O2, and release of AZM could effectively reduce the bacterial infection and promote the wounds healing. This accelerated anti-bacteria and wound healing process are mainly due to the destruction of bacterial biofilm and the acceleration of vascular regeneration by GOX-HMSN-AZM. More importantly, the biosafety of the composite nanoparticles was verified during the therapy. This work provides a meaningful inspiration for the rational design of intelligent nanocomposites to realize efficient therapeutic modalities for chronic diabetic wounds, which has profound significance for clinical application.In summary, we successfully developed multifunctional GOX-HMSN-AZM nanoparticles for the treatment of chronic diabetic wounds. Owing to the unique catalytic capability of GOX, GOX-HMSN-AZM could effectively ameliorate the hyperglycemia environment around the wound and produce HSupplementary figures and table.Click here for additional data file.


The burden of pancreatic ductal adenocarcinoma (PDAC) increases with rising incidence, yet 5-year overall survival remains poor at 17%. Routine comprehensive genomic profiling of PDAC only finds 2.5% of patients who may benefit and receive matched targeted therapy. Protein arginine methyltransferase 5 (PRMT5) as an anti-cancer target has gained significant interest in recent years and high levels of PRMT5 protein are associated with worse survival outcomes across multiple cancer types. Inhibition of PRMT5 in pre-clinical models can lead to cancer growth inhibition. However, PRMT5 is involved in multiple cellular processes, thus determining its mechanism of action is challenging. While past reviews on PRMT5 have focused on its role in diverse cellular processes and past research studies have focused mainly on haematological malignancies and glioblastoma, this review provides an overview of the possible biological mechanisms of action of PRMT5 inhibition and its potential as a treatment in pancreatic cancer.The overall survival of pancreatic ductal adenocarcinoma (PDAC) remains poor and its incidence is rising. Targetable mutations in PDAC are rare, thus novel therapeutic approaches are needed. Protein arginine methyltransferase 5 (PRMT5) overexpression is associated with worse survival and inhibition of PRMT5 results in decreased cancer growth across multiple cancers, including PDAC. Emerging evidence also suggests that altered RNA processing is a driver in PDAC tumorigenesis and creates a partial dependency on this process. PRMT5 inhibition induces altered splicing and this vulnerability can be exploited as a novel therapeutic approach. Three possible biological pathways underpinning the action of PRMT5 inhibitors are discussed; c-Myc regulation appears central to its action in the PDAC setting. Whilst homozygous MTAP deletion and symmetrical dimethylation levels are associated with increased sensitivity to PRMT5 inhibition, neither measure robustly predicts its growth inhibitory response. The immunomodulatory effect of PRMT5 inhibitors on the tumour microenvironment will also be discussed, based on emerging evidence that PDAC stroma has a significant bearing on disease behaviour and response to therapy. Lastly, with the above caveats in mind, current knowledge gaps and the implications and rationales for PRMT5 inhibitor development in PDAC will be explored. Since the 1990s, the global pancreatic ductal adenocarcinoma (PDAC) incidences and deaths across 185 countries have increased by 2.3 fold, partly coinciding with the increasing prevalence of diabetes and obesity ,5,6. ComNonetheless, genomic profiling has uncovered distinct PDAC molecular subgroups with their own metabolic and prognostic differences that may explain the clinical heterogeneity in PDAC outcomes ,26,27,28Past genomic profiling efforts have also revealed that alteration in RNA processing, with recurrent mutations in splicing factors SF3B1, RBM10 and U2AF1, is one of the hallmark drivers of PDAC tumorigenesis . UnfortuNumerous reports demonstrate that high protein arginine methyltransferase 5 (PRMT5) expression is associated with a worse prognosis across multiple cancer types, including PDAC. Inhibition of PRMT5 in preclinical models leads to reduced tumour growth ,41,42,43In the best-case scenario, patients newly diagnosed with PDAC undergo both surgical resection and complete six months of modified FOLFIRINOX adjuvant chemotherapy to achieve a median OS of 54 months and 5 year OS of 43% ,53. SurvThe U.S. Food and Drug Administration (FDA) has approved olaparib for PDAC patients who are carriers of germline BRCA mutations. However, olaparib only confers 3.8 months improvement in progression-free survival (PFS) without any OS benefit, nor improvement in quality of life compared to a truncated course of chemotherapy ,59. AlthProtein arginine methylation is an important post-translational modification that involves protein arginine methyltransferases (PRMTs) catalysing the transfer of methyl group from S-Adenosylmethionine (SAM) to arginine residues on histone and non-histone proteins . The addDuring the past 10 years, PRMT5 has emerged as a potential therapeutic target in multiple solid tumours ,40,41,42The effect of PRMT5 inhibition on PDAC growth is also intriguing as this may be mediated by its effect on alternative splicing and may represent a novel therapeutic class . AberranThe translation of PRMT5 into a therapeutic target has been slow due to challenges in identifying the relevant mechanism of action, since PRMT5 is involved in a plethora of pathways and multilevel control of gene and protein expression  112]. F. F112]. Although PRMT5 has differing roles in many cellular processes, three characterised pathways are thought to mediate the growth inhibition effect seen with PRMT5 inhibitors. Depending on which biological pathway is pertinent to the growth inhibitory action of PRMT5, different PDAC molecular subtypes may have different responses to PRMT5 inhibitors.One of the best-characterised effects of PRMT5 inhibition is the upregulation of p53 activity through alternative splicing of MDM4 . PRMT5 iIn PDAC cell lines, PRMT5 indirectly induces autophosphorylation of tyrosine residues Y1068 and Y1172 on epidermal growth factor receptor (EGFR) . It is aIf the PRMT5 inhibitory effect is mediated via the GSK3β axis, its effectiveness may be limited to a subset of PDAC based on the effects seen with direct GSK3β inhibitor. Despite KRAS mutation being the main driver for PDAC tumourigenesis, only a subset of KRAS mutant shows dependency where siRNA inhibition of KRAS induces apoptosis and caspase-mediated death. Similarly, GSK3β inhibition selectively causes apoptosis in these “KRAS dependent” PDAC . More reThe ability of PRMT5 to regulate pathways at multiple levels further adds to the complexity in deciphering its primary mechanism of action. For example, PRMT5 can modulate the NFkβ pathway via (1) regulation of GSK3β and a subsequent inhibitory effect on IKK, (2) direct methylation of p65 subunit l, preventing translocation of NFkβ into the nucleus  Figure , and lasFBXW7 is a potent tumour suppressor gene that functions as part of the Skp1-Cullin1-F-box (SCF) ubiquitin ligase complex that targets major oncoproteins, including Notch-1 , c-Myc, Of note, the degradation of MYC requires priming by ERK-dependent phosphorylation at serine 62 followed by GSK3B mediated phosphorylation at threonine 58 before MYC can interact with FBXW7 for polyubiquitination mediated degradation . TherefoBesides identifying the alterations in signalling in different biological pathways, examining how alternative splicing is perturbed may shed light on PRMT5 inhibitors’ mechanism of action as the two are seemingly linked. PRMT5 inhibition leads to thousands of alternative splicing events (ASEs) with enrichment in genes in RNA processing . HoweverCuriously, the addition of PRMT1 to PRMT5 inhibition in a pancreatic cancer cell line induces a synergistic increase in >2400 novel ASEs compared with PRMT5 or PRMT1 inhibitor alone. Conversely, only approximately 220 ASEs were commonly induced by both inhibitors individually . Dual coSimilarly, in acute myeloid leukaemia (AML) cell lines, combination PRMT1 and PRMT5 inhibition also result in synergistic increases in novel ASEs . The vasReliance on published gene expression data and splicing analysis following PRMT5 treatment at a single timepoint limits the potential to assess the biological effects of PRMT5 inhibitors comprehensively. Detained introns from alternative splicing impair mRNA transport out of the nucleus, thus reducing mRNA translation Step 6,  138,149149. PRMTWith the pleiotropic effects of PRMT5 and the multitude of possible mechanisms of action of PRMT5 inhibitors, a predictive biomarker is ever more critical to better select appropriate patients who will benefit from targeted PRMT5 inhibition. Unfortunately, currently proposed biomarkers of sensitivity to PRMT5 inhibition are inadequate at predicting the growth inhibitory effect and thus preclude their incorporation into clinical practice even though they offer mechanistic insights.CDKN2A gene on chromosome 9 [The most commonly cited biomarker of sensitivity is the homozygous deletion of the methylthioadenosine phosphorylase (MTAP) gene. This occurs in 25% of PDAC, owing to the MTAP gene’s proximity to the frequently deleted mosome 9 ,151. Biomosome 9 ,155,156.mosome 9 . Of notemosome 9 . This mamosome 9 . Therefomosome 9 . Newer gmosome 9 ,158.Although the relevance of MTAP appears somewhat promising based on pancreatic organoid data alone, examination across a wider number of cell lines suggests MTAP status predictiveness is less robust. Further examination across 11 pancreatic cell lines and a broader 64 cell line panel revealed no correlation between response to PRMT5 inhibition and MTAP status ,156. EquThe other common “biomarker” used in the field is measuring the SDMA level, which is a functional measure of PRMT5 specific methylation activity rather than its clinical/biological effect ,162. In What is less discussed outside of the haematological context is the relevance of recurrent mutation in splicing factors that may confer increased sensitivity to PRMT5 inhibition, arising from their increased susceptibility to further perturbation of altered splicing ,165,166.With limited targetable mutations in PDAC, attention had turned to target the characteristic desmoplastic stroma that supports PDAC tumorigenesis. Traditionally, the desmoplastic stroma was thought to confer chemotherapy resistance which could be reversed with sonic hedgehog inhibitors in pre-clinical models. However, a clinical trial of this inhibitor led to the paradoxical acceleration of PDAC growth . The sonFurther evidence of PDAC and stroma interaction comes from the observation that mutant p53 PDAC can educate CAFs. These “educated” CAFs can instruct the more indolent wildtype p53 PDAC to adopt a more invasive phenotype, either directly via facilitation of invasion, or indirectly by increasing perlecan/HSPG2 secretion. ,171. In + T cells [PRMT5 inhibition may enhance immunogenicity in PDAC, based on the predicted impact on the PDAC microenvironment and effects reported in other cancers. Outside of the microsatellite unstable setting, immunotherapy trials in PDAC have all been negative ,174, exc T cells . In addi T cells . In mela T cells ,49. ThesIn contrast, in human peripheral blood mononuclear cells, PRMT5 inhibition reduces CD8+ T cell proliferation via the MDM4/p53 axis and reduces metabolic activity (glycolysis and fatty acid oxidation) via inhibition of AKT/mTOR pathways . PRMT5 iIn the era of precision oncology, melanoma and lung cancer have recently seen giant strides in improving survival outcomes, with the advent of immunotherapy and the identification of subgroups amenable to targeted therapies. Advanced PDAC continues to have a poor prognosis with a median survival of fewer than 12 months  and withThe scientific challenge is that PRMT5 has multiple cellular functions and regulates signalling at multiple levels within a pathway and via differing mechanisms. Nonetheless, the regulation of c-Myc expression thus far seems to be central to mediating the inhibition of cancer growth. The apparent redundancy of inhibitory actions that PRMT5 has on c-Myc stability is not well understood. However, PRMT5 inhibition may result in a longer duration of disease control due to concurrent inhibition of multiple parallel signalling pathways. This potentially reduces the rate of adaptive resistance development that is seen with other targeted therapies. Lack of a singular mechanism of action, as seen with oncogenes, and the varied mechanisms across different cancers suggest that its mechanism of action is likely unique to a particular cancer type. However, given that the response to PRMT5 inhibitor therapy is partially dependent on molecular contexts such as MTAP deletion or altered splicing as seen in glioblastoma, its mechanism may not be tissue-specific but molecularly defined. Therefore, understanding the alternative splicing landscape in PDAC is likely to be important in defining the use of PRMT5 inhibitors for this disease. With the poor response rate seen in early phase trials, there is a strong need for a better predictive response biomarker, as the current “biomarker” of SDMA levels are functional measures of PRMT5 activity but not necessarily representative of its biological effect .To advance the translation of PRMT5 inhibitors to a clinical trial in PDAC, better elucidation of the PRMT5 inhibitor’s mechanism of action and/or identification of appropriate predictive response biomarkers will be paramount. Firstly, acknowledging the plethora of potential actions of PRMT5, unbiased and systematic assessment of which genes PRMT5 inhibition affects will be beneficial to narrow down biological pathways of relevance. Examples of these approaches would include whole-genome CRISPR screens to identify the essential genes required for response to PRMT5 inhibition and transcriptomic assessment of differential gene expression and splicing changes. However, reliance on conventional knock-out screens or transcriptomic analysis alone is insufficient to elucidate the multiplicity of effects that PRMT5 can have over gene and protein expression. Therefore, multi-omic strategies, including proteomic, are likely needed to comprehensively ascertain the relative importance and interdependency of different mechanisms that PRMT5 can concurrently modulate, such as epigenetic, translational efficiency, RNA transport, protein stability and crosstalk with phosphorylation and ubiquitination processes  Figure . NonetheLastly, with the emerging evidence suggesting the importance of stroma in determining PDAC behaviour and the dynamic nature of the stroma, PRMT5 inhibitor development in PDAC must be considered in this context. It remains to be further elucidated whether the pro-immunomodulatory effect PRMT5 inhibition has on the microenvironment is sufficient to override the inhibition of immune cells in the peripheral system. This is particularly important in the PDAC setting, where there are insufficient numbers of activated immune cells to orchestrate an immune response, perhaps explaining the negative findings in immunotherapy trials to date.With all these caveats, PRMT5 remains a potential target for PDAC, and its ability to modulate alternative splicing provides a novel therapeutic class for the treatment of this disease. Biologically, with PRMT5 having multiple control points within and across differing signalling pathways, this agent may be less likely to induce tumour adaptation and resistance, as seen in many targeted therapies . CurrentIn view of limited targeted therapy options available and continued poor survival with conventional cytotoxic chemotherapy, novel therapeutic options are needed to improve the survival outcomes of patients with advanced PDAC significantly. PRMT5 inhibition poses an interesting target given its effects on alternative splicing, a known hallmark driver in PDAC pathogenesis. The pleiotropic effects of PRMT5 inhibition may depend on the molecular context and thus differ from conventional oncogene inhibitor therapy. While this poses challenges to understanding its mechanism of action and appropriate patient selection, PRMT5 inhibition may result in less chance of therapy resistance, due to redundancy in its actions on either the same or complimentary signaling pathways. Therefore, PRMT5 inhibition is a note worthy target to further develop for the treatment of PDAC.


Lamins are intermediate filaments that play a crucial role in sensing mechanical strain in the nucleus of cells. β-catenin and megakaryoblastic leukemia-1 (MKL1) are critical signaling molecules that need to be translocated to the nucleus for their transcription in response to mechanical strain that induces osteogenesis. However, the exact molecular mechanism behind the translocation of these molecules has not been fully investigated. This study used 10% cyclic strain to induce osteogenesis in the murine osteoblast precursor cell line (MC3T3). The translocation of β-catenin and MKL1 was studied by performing knockdown and overexpression of lamin A/C (LMNA). Cyclic strain increased the expression of osteogenic markers such as alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), and enhanced ALP staining after seven days of incubation. Resultantly, MKL1 and β-catenin were translocated in the nucleus from the cytoplasm during the stress-induced osteogenic process. Knockdown of LMNA decreased the accumulation of MKL1 and β-catenin in the nucleus, whereas overexpression of LMNA increased the translocation of these molecules. In conclusion, our study indicates that both MKL1 and β-catenin molecules are dependent on the expression of LMNA during strain-induced osteogenesis. The nuclear lamina is a type VI intermediate filaments meshwork found inside the nuclear envelope. In addition to its role as a structural barrier between cytoplasm and nucleus, it plays an essential part in several cellular processes. Two functions are usually assigned to the nuclear lamins: 1) it provides shape and structural support to the nucleus; 2) it regulates cell signaling and chromatin remodeling. Recently, a few studies have suggested the role of nuclear lamin in sensing mechanical forces and acting as a mediator to generate a mechanical response to adapt to the changing microenvironment [ it provi it regulTwo isoforms of nuclear lamins, generated by splicing lamin A/C (LMNA) genes, i.e., lamin type A and C, are highly expressed in differentiated cells . SeveralThe role of LMNA in osteogenesis has attracted attention, since it plays a significant role in laminopathies ,12. A stMechanical strain plays an essential role in regulating bone regeneration, since increased mechanical strain causes osteoblast proliferation and differentiation ,18. One Previous reports have established that Wnt/β-catenin and MKL1/SRF pathways are regulatory factors for osteogenesis ,38,39. βMC3T3-E1 cells were cultured on FlexCells culture plates, and 10% cyclic strain was applied on the 1st, 4th, and 7th days A. WesterFurthermore, lamin A/C protein expression peaked at day seven compared to control. The immunofluorescence expression of LMNA also became prominent on the 4th and 7th days after applying cyclic strain E. SimilaAfter investigating the protein expression during the cyclic strain, we observed the translocation of β-catenin and MKL1 signaling molecules in the cytoplasm and nucleus. A cytoplasmic and nuclear extraction kit was used, wherein samples were collected immediately after applying strain. The result showed that MKL1 gradually decreased in the cytoplasm and reached its lowest level on the 7th day. Conversely, translocation in the nucleus increased slowly and reached to peak on the 7th day. Similarly, β-catenin also decreased gradually over time in the cytoplasm, and its accumulation significantly increased in the nucleus by following the same trend A,B. ImmuAs we observed increased LMNA expression and increased nuclear accumulation of MKL1 and β-catenin, we speculated that LMNA might influence the translocation of these molecules during the process. We then knocked down and overexpressed LMNA to investigate its effect on the translocation of signaling molecules. Sh plasmid and lentivirus were used for the knockdown and the overexpression of LMNA, respectively. Fluorescence microscopy was used to observe green fluorescent protein (GFP), which showed that transfection efficiency was above 80% A. Both kThe cyclic strain was applied for seven days, and the cytoplasmic and nuclear protein were collected on the 7th day. Knocking down LMNA reduced total MKL1, RUNX2 expression significantly; however, its effect on β-catenin was insignificant A,B. It iWe then investigated whether increased LMNA expression causes increased nuclear accumulation. LMNA overexpression caused a significant rise in the expression of β-catenin, RUNX2, and MKL1. Besides, both MKL1 and β-catenin were prominently expressed in the nucleus. Moreover, immunofluorescence confirmed the Western blot results, as MKL1 and β-catenin were highly expressed in the nucleus in overexpressed LMNA cells A,B. ThesMechanotransduction initiates the movement of mechanosensitive molecules and transcription factors in the nucleus during and after the mechanical force ,48. The The cyclic mechanical strain has been reported to induce osteogenesis in osteoblast cell lines and mesenchymal stem cells ,53. In oTranslocation of β-catenin and MKL1 in the nucleus reached a peak on the 4th and 7th days. Our study demonstrated that β-catenin increased in the cytoplasm on the first day and then decreased gradually, which is supported by the fact that β-catenin is a mechanosensitive signaling molecule bound to cadherin mechanosensor. After the application of strain, β-catenin is released free from cadherin in the cytoplasm . The traIn our study, alizarin staining was more evident in LMNA overexpressed cells, indicating more osteogenesis in LMNA overexpressed cells than in LMNA knockdown and control. LMNA knockdown decreased the MKL1, RUNX2 expression, and its overexpression increased MKL1, β-catenin, and RUNX2 expression. This could be supported by the fact that MKL1 decreased its response in MSCs when cultured on the soft substrate, and its expression increased when cultured on the stiff substrate . In our In summary, we present the first evidence that translocation of MKL1 and β-catenin is dependent on LMNA under the cyclic strain’s induced osteogenesis. However, we think there are some limitations in our study, i.e., firstly, the study duration is seven days, which could only tell us about the initial stage of mechanical induced osteogenesis. Secondly, we observed the MKL1 and β-catenin translocation immediately after applying strain; however, translocation could also be studied on different time scales after two hours or four hours, which helps us give more insight into the translocation of transcription factors. Overall, these data suggest that MKL1 and β-catenin are dependent on LMNA for their translocation in the nucleus under the mechanical strain. Future studies will focus on cross-talk between MKL1 and β-catenin under different time durations and frequencies of mechanical strain.2 atmosphere. The medium was supplemented with 10% FBS (Gibco) and 1% penicillin and streptomycin (Gibco). The medium was changed regularly every second day to maintain the working conditions.MC3T3-E1 cells were purchased from Chinese Academy Sciences cell back bank . MC3T3-E1 cells were cultured in MEM alpha  at 37 °C in a 5% CO5 cells on specialized collagen-coated silicone membrane plates . The cyclic strain was applied after 48 h of culturing MC3T3-E1 cells on the plate. Flexcell FX-5000 system   A was usePhosphate buffer saline (PBS) was used to wash the cells, and then 4% paraformaldehyde was used to fix the cells. PBS was used to wash again, and ALP staining was used . Cells were incubated for 30 min at 37 °C and then washed twice with PBS. An optical microscope was used to observe the cells after washing with PBS.Culture medium was removed from the plates, and PBS was used to wash the cells twice. Fixation was carried out using 4% paraformaldehyde followed by washing of the cells with PBS. Cells were incubated with alizarin staining  for 10 min and then washed again with PBS two times. Stained cells were observed under the microscope .® Statistics 20 software.Followed by washing with PBS three times, cells were fixed with 4% paraformaldehyde  for 10 min at room temperature. PBS was used to wash these cells after fixation, and 0.1% Triton X-100 was used for 5 min. Then, blocking was performed by 5% bovine albumin serum for 30 min at room temperature. Cells were incubated overnight at 4 °C with primary antibodies , Rabbit anti-MKL1 , rabbit anti-β-catenin , and Phalloidin Red-594 ). Cells were again washed three times, and the second antibodies (AlexaFluor488 goat anti-mouse (1:500), AlexaFluor488 goat anti-rabbit (1:500), AlexaFluor568 goat anti-mouse (1:500), AlexaFluor568 goat anti-rabbit (1:500), ) were added. Cells were incubated for 1 h in the dark. These cells were imaged under confocal microscopy, and the data were analyzed using the SPSSAfter applying mechanical strain, cells were harvested immediately on days 1, 4, and 7. Cytoplasmic Protein Extraction Kit  was used, and protein extraction was done according to the manufacturer’s instructions. According to the provided protocol, the cytoplasmic protein was extracted first, followed by the nuclear protein extraction.After harvesting and washing the cells, the protein was extracted by preparing immunoprecipitation lysis buffer using the instruction given in the whole protein extraction kit . A total of 10% SDS polyacrylamide gel electrophoresis was used to separate the protein and then transfer it onto polyvinylidene difluoride (PVDF) membranes. After the transfer, blocking was carried out by 5% skim milk for one hour. All membranes were incubated at 4 °C overnight in the primary antibody mouse anti-Lamin A/C , Rabbit anti-MKL1 (1:1000 Abcam ab49311), rabbit anti-β-catenin  mouse anti-ALP , rabbit anti-RUNX2 , and rabbit anti-GAPDH  . After that, membranes were washed and incubated with a second antibody at room temperature for two hours. Enhanced chemiluminescence (ECL) chromogenic substrate was used to observe the band.Trizol  was used to extract the total RNA from the cells following the instructions given by the company. RevertAid First Strand cDNA Synthesis Kit  was used to reverse-transcribed RNA into cDNA. ABI StepOne Plus System  was used to perform a quantitative real-time polymerase chain reaction (qPCR). Following primer sequences were used to express LMNA, β-catenin, RUNX2, ALP, MKL1 Lentivirus for negative control (CON335) and LV-LMNA (66639) constructed by Genechem  were used to trigger overexpression of LMNA. First, the optimal condition was selected, including optimal concentration, volume, treatment duration, and the number of cells using multiple treatments. After that, virus dilution in the fresh medium according to transfected cell density ratio and virus efficiency enhancer were added. Firstly, approximately 7000 cells/cm2 were cultures in 6-well culture plates. When the confluence reached 20 to 30 percent, we transfected the virus into the cells using the cell multiplicity of infection (MOI) and virus titer. The medium was replaced with a growth medium after 12 h, and expression efficiency was observed under an Olympus microscope .Cells were grown in 6-well plates, and the growth medium was supplemented until it reached the confluence of 70%. LMNA Sh-RNA plasmid  were diluted in transfection medium  and incubated for 30 min at room temperature. Cells were washed with transfection medium, and plasmid was inoculated for 7 h. The medium was aspirated after 7 h, and the growth medium was transferred into it. Western blot and RT-qPCR experiments were performed to confirm the knockdown.


Nobiletin is a natural compound with anticancer activity; however, the mechanism is not clear.  The inhibitory effect of nobiletin on non-small-cell lung cancer (NSCLC) cells was examined using soft agar, Transwell, and apoptosis analyses. Cancer stemness was measured by sphere assay. Genes and miRNAs regulated by nobiletin were identified by whole-genome sequencing. Protein levels were detected by western blot and immunofluorescence assays. β-catenin signaling, including NKD1, AXIN2, and WIF1, while it inhibited the expression of β-catenin and its downstream genes, including c-Myc, c-Jun, and cyclin D1. Furthermore, we identified that GN inhibits miR-15-5p expression in NSCLC cells and that NKD1, AXIN2, and WIF1 are the target genes of miR-15-5p.  Nobiletin significantly inhibited NSCLC cell colony formation and sphere formation and induced apoptosis. Nobiletin upregulated negative regulators of WNT/β-catenin signaling in NSCLC. Nobiletin has a strong inhibitory effect on NSCLC, and nobiletin plays an anticancer role by inhibiting miR-15-5p/ Citrus unshiu (Satsuma mandarin) and Citrus sinensis. Studies show that nobiletin exhibits an anticancer role in various cancers, including skin, prostate, colon, and breast cancer [Lung cancer is the most frequently diagnosed malignancy and the leading cause of cancer-related mortality worldwide –3. Non-st cancer –18. Howeβ-catenin signaling is a well-studied oncogenic pathway that plays an important role in the development and progression of various cancers. For example, there have been some reports that WNT/β-catenin might enhance the motility of malignant cells and tumor invasion, such as in breast cancer, melanoma, and gastric cancer [β-catenin signaling pathway [β-catenin signaling is a useful strategy for cancer treatment.WNT/c cancer –23. As a pathway –3. In faβ-catenin signaling by upregulating the expression levels of WIF1, AXIN2, and NKD1. We also identified that nobiletin inhibits the expression of WIF1, AXIN2, and NKD1 by inhibiting miR-15-5p in NSCLC cells.In this study, we demonstrated that nobiletin significantly inhibits NSCLC growth, metastasis, and stemness and stimulates NSCLC cell apoptosis. In addition, we demonstrated that nobiletin plays its role by inactivating Wnt/Bicinchoninic acid (BCA) assay reagent was purchased from Bio-Rad , Lipofectamine was purchased from Invitrogen , the ECL (enhanced chemiluminescence) kit was purchased from Amersham Pharmacia , and the Immobilon-p membrane was purchased from Millipore . RPMI  1640 medium, fetal bovine serum (FBS), TryPLE-Express enzyme, and Hank's balanced salt solution were obtained from Gibco .2 humidified atmosphere at 37°C. The cells were then treated with 2.5 µM nobiletin for 24 hours and then subjected to a soft agar assay. After 10 days, the cell cultures were treated with 10 µL of CCK-8 solution for 2 hours and incubated at 37°C for the determination of cell viability. A microplate reader was used to obtain the optical density (OD) at 40 nm. This research was approved by the Institutional Review Board of Chuncheon Sacred Heart Hospital.Cell lines H460 and A549 were obtained from ATCC , and these cells were cultured in DMEM  supplemented with 10% FBS  in a 5% COFor the cell viability assay, H460 and H549 cells were seeded in 96-well plates at a density of 5000/well. The next day, the cells were treated with nobiletin or PBS for 48 h, and cell viability was measured using a Cell Counting Kit-8 . For the soft agar assay, cells were treated with the indicated concentration of nobiletin for 48 h, and then, cells were resuspended in 0.5 mL of 0.35% agar (Sigma) in the growth medium at a density of 5000 cells/well in 6-well plates. The agar-cell mixture was plated on the top of a solid layer of 0.8% agar in the growth medium. Colonies were counted 14 days later.4 cells in the serum-free medium were plated into the top chamber with or without gel coating. The cell growth medium with 10% FBS was added under the chamber. After 24 h, cells on the upper sides of the inserts were removed using cotton, and cells on the surface of the bottom sides of the inserts were fixed with 4% paraformaldehyde, followed by staining with 0.5% crystal violet. The cells in five random microscopic fields were photographed using an inverted phase-contrast microscope and counted.Cell migration and invasion assays were performed using a Transwell chamber. Cells were treated with the indicated concentration of nobiletin for 48 h. Then, the cells were resuspended, and 2 × 104 cells per ml of the sphere-forming medium consisting of a 1 : 1 mixture of high-glucose DMEM and Ham's Nutrient Mixture F-12  supplemented with 20 ng/ml EGF and 20 ng/ml recombinant FGFb, 1X Insulin-Transferrin-Selenium , 4 mg/ml bovine serum albumin , 2 mM glutamine (Euroclone), 50 U/ml penicillin, and 0.05 mg/ml streptomycin (Euroclone). After 6–7 days of growth, spheres were counted.Cells were treated with the indicated concentration of nobiletin for 48 h, and then, a sphere formation assay was carried out as described in previous studies –20, 24. The luciferase reporter assay was carried out as described by Fend .Western blot and RT-PCR analyses were carried out as described in previous studies –24. For P < 0.05) between groups were analyzed by Student's t-test using SAS statistical software version 6.12 (SAS Institute).All data are presented as the mean ± standard deviation (SD), and significant differences  and H460 (P < 0.05) NSCLC cells compared to their control group. Consistently, migration assays also showed that nobiletin significantly inhibited the migration abilities of both A549 and H460 cells (Figures Metastasis is a poor prognostic factor of NSCLC. Therefore, we investigated the effect of nobiletin on the invasion and migration of A549 and H460 cells. Transwell chamber assays were conducted to evaluate the effects of nobiletin on invasion and migration. As shown in Figures  Figures , suggestβ-catenin signaling (β-catenin signaling, including NKD1, AXIN2, and WIF1 while inhibiting positive regulators of WNT/β-catenin signaling, including WNT6 and Jun (β-catenin signaling.To investigate the anticancer molecular mechanism of nobiletin in NSCLC, A549 and H460 cells were treated with nobiletin for 48 hours and then subjected to whole-genome sequencing. Nobiletin treatment altered the expression levels of many genes in both NSCLC cell lines . In addiignaling . In fact and Jun . Consist and Jun . Togetheβ-catenin signaling, NSCLC cells were treated with nobiletin and then subjected to sphere formation assays, and the expression levels of marker proteins of cancer stemness and WNT/β-catenin signaling were detected. Our results showed that nobiletin significantly inhibited sphere formation of NSCLC cells (β-catenin levels (Figures β-catenin signaling (β-catenin signaling pathway in NSCLC.To investigate whether nobiletin is directly involved in the inhibition of cancer stemness and WNT/LC cells  and cancLC cells . In addi Figures  and downignaling . These rβ-catenin signaling, we performed miRNA sequencing using nobiletin-treated NSCLC cells and their control cells because previous studies showed that miRNAs were involved in the regulation of WNT/β-catenin signaling by targeting their regulators [β-catenin signaling, including NKD1, AXIN2, and WIF1 (To investigate the inhibitory mechanism of nobiletin on WNT/gulators . miRNA sand WIF1 . First, and WIF1 . In addiand WIF1 . Notablyand WIF1  significand WIF1  and protand WIF1 .β-catenin signaling. As shown in Figures β-catenin expression level, while overexpression of miR-15-5p increased the β-catenin expression level. Notably, overexpression of miR-15-5p blocked nobiletin-induced inhibition of β-catenin expression (β-catenin signaling through inhibition of miR-15-5p in NSCLC.Next, we investigated whether miR-15-5p was involved in nobiletin-induced inhibition of WNT/pression , suggestIn recent decades, natural products have received widespread attention as anticancer drugs. They have anticancer effects and fewer side effects –4. Howevβ-catenin signaling is one of them [β-catenin signaling, suggesting that nobiletin exerts its anticancer effects by inhibiting WNT/β-catenin signaling-regulated cancer stemness and EMT.Cancer stemness is a key factor for cancer progression. Studies show that increased cancer stemness stimulates cancer cell resistance to chemotherapy and enhances cancer cell invasiveness, thereby contributing to cancer progression , and inc of them , 31. Herβ-catenin signaling was inactivated by nobiletin. WNT/β-catenin signaling is often hyperactivated in cancers by the downregulation of negative regulators, including NSCLC [β-catenin signaling pathway is promoted by dysregulated miRNA expression in cancer [β-catenin signaling, such as NKD1, AXIN2, and WIF1, which inactivate WNT/β-catenin signaling.Here, we also demonstrated that WNT/ng NSCLC . In addin cancer . Interesn cancer –36. In tβ-catenin signaling pathway in NSCLC. Nobiletin inhibited miR-15-5p expression, thereby increasing negative regulators of the WNT/β-catenin signaling pathway and ultimately inactivating the WNT/β-catenin signaling pathway. Our results shed light on the mechanism of the anticancer effect of nobiletin and provide more inspiration for physicians in treating patients with NSCLC.In conclusion, nobiletin inhibits NSCLC by inhibiting cancer stemness and EMT by inactivating the WNT/


Using an in vitro skin culture model and an in vivo skin wound model, our study further reinforced the critical role of ROS in inducing EMT through SOX2 expression and subsequent activation of Wnt/β-catenin, allowing for rapid reepithelialization of the wound area. Taken together, our findings reveal a previously unknown mechanism by which hypoxia promotes wound healing by promoting reepithelialization through the production of ROS, inducing keratinocyte EMT and migration via the enhancement of SOX2 and activation of Wnt/β-catenin.We previously showed that wound-induced hypoxia is related to keratinocyte migration. The ability of keratinocytes within wound healing to undergo epithelial to mesenchymal transition (EMT) contributes significantly to the acquisition of migratory properties. However, the effect of hypoxia on keratinocyte EMT on wound healing and the potential mechanism are poorly documented. This study first demonstrated that reactive oxygen species (ROS) appear to be an essential signalling mediator in keratinocytes with increased EMT and migration subjected to hypoxic conditions. Next, we showed that the expression of sex-determining region Y-box 2 (SOX2), a stemness-associated molecule, is ROS-dependent under hypoxia and that SOX2 inhibition in keratinocytes dramatically prevented hypoxia-induced EMT and migration. In addition,  Mammalian cutaneous wound healing is a well-coordinated process that involves three consecutive but overlapping phases: inflammation, reepithelialization, and maturation. During the wound reepithelialization phase, keratinocytes migrate into the wound site, proliferate, and differentiate to reestablish barrier function and homeostasis of the skin. Dysfunctional keratinocyte migration is an important cause of unsatisfactory wound repair , 2. Rema2 owing to the loss of vascular structure and heightened oxygen consumption [2 tension is a vital component of the epidermal microenvironment that contributes to skin homeostasis and development. Our previous studies have shown that wound-induced hypoxia (2% O2) enhances keratinocyte migration [Following acute injury, the epidermis on the wound site is normally deprived of Osumption . Furtherigration , but theigration . Since kHypoxia induces reductive carboxylation to adapt cells to a low oxygen environment, which was shown to increase reactive oxygen species (ROS) generation . Many stThis study was aimed at determining the relationship between hypoxia and keratinocyte EMT and deciphering the underlying mechanisms. Additionally, we investigated whether ROS were involved in the regulation of EMT induced in hypoxic keratinocyte cells.All animal studies followed the NIH Guide for the Care and Use of Laboratory Animals  and were approved by the research Ethics Committee of Shenzhen People's Hospital, China.2 and 95% humidity at 37°C. Skin biopsies were derived from the dorsal skin of newborn C57BL/6 mice . Skin organ culture was performed as described in our previous report [Immortalized human keratinocyte HaCaT cells were purchased from the Cell Bank of the Chinese Academy of Sciences , originally from American Type Culture Collection . The cells were cultured in a modified Eagle's medium (Gibco) containing 10% foetal bovine serum (FBS) and 1% penicillin and streptomycin (Gibco). Cells were maintained under standard cell culture conditions of 5% CO2, 5% CO2, and 93% N2 were produced utilizing an oxygen control incubator (Smartor 118), which maintained a low O2 level under constant nitrogen flow at 37°C. The ROS scavenger NAC , the β-catenin inhibitor XAV939 , and acriflavine (ACF)  were added to the cultures and incubated at 37°C during hypoxia treatment.Hypoxic conditions of 2% Oβ-catenin , LEF1 , LGR5 , and β-actin . Western blot analyses were performed as described in our previous report [http://rsb.info.nih.gov/ij/).Whole-keratinocyte extracts and mouse skin specimens were first obtained for Western blot analyses and centrifuged at 14,000 rpm for 15 min at 4°C. The supernatants were then prepared, and protein concentrations were determined with a Bradford Protein Quantification Kit . The protein extracts were separated by SDS-PAGE and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore). The membranes were incubated overnight at 4°C with corresponding primary antibodies and secondary antibodies and visualized by the ChemiDoc XRS system (Bio-Rad Laboratories). Primary antibodies against the following were used to detect protein expression: HIF1 , vimentin , E-cadherin , N-cadherin , sex-determining region Y-box 2 (SOX2) , Slug , s report . QuantifAn inverted phase-contrast microscope was used to obtain representative images from HaCaT cells subjected to hypoxia treatment or normoxia treatment.After treatment, the samples (cells cultured on glass coverslips and frozen mouse skin sections) were fixed in 4% paraformaldehyde for 20 min. The samples were incubated with primary antibodies at 4°C overnight and washed three times with phosphate-buffered saline (PBS). Next, the samples were stained with fluorescent secondary antibodies for 1 h at 37°C. Primary antibodies against the following were used: vimentin , E-cadherin , N-cadherin , SOX2 , and Slug . The following secondary antibodies were used: FITC-labelled goat anti-rabbit IgG  and DyLight 594-labelled goat anti-mouse IgG . The nuclei were then counterstained with DAPI (Abcam) before imaging. IF images were captured using fluorescence microscopy .μl plastic pipets. At 12 hours after wounding, cells that migrated into the cell-free area were monitored with an inverted light microscope . Cell migratory ability was defined as the relative migrating area (%), which was measured using NIH ImageJ software.HaCaT cells were plated on 6-well plates. Cell monolayers were scratched with 200 μM) for 20 min at 37°C. The samples were quickly washed twice with PBS to remove the extra dye and then detected and imaged under a fluorescence microscope. The fluorescence images were observed with a microplate reader, and fluorescence was measured with a fluorescence microplate reader  with an excitation wavelength of 488 nm and emission wavelength of 525 nm for DCFH-DA.After various treatments, ROS levels were detected with a fluorescence microscope using the redox-sensitive fluorescent dye 2′,7′dichlorofluorescein . Cells and frozen skin sections were incubated with DCFH-DA . The amount of RNA extracted was measured with OD260/OD280 ratios between 1.9 and 2.1, and the total isolated RNA was reverse transcribed into cDNA using the PrimeScript RT reagent Kit . Real-time PCR was performed using TB Green Premix Ex Taq  on a StepOnePlus quantitative PCR system . The primer sequences used for qRT-PCR are listed in Supplementary Table On the day prior to transfection, cells were plated to the required cell density (at least 70% confluency). Small interfering RNAs (siRNAs) specific for SOX2 and the corresponding scrambled siRNA  were individually diluted in Opti-MEM (Life Technologies) and incubated for 5 min at room temperature. The diluted siRNAs were added to diluted Lipofectamine 2000  and further incubated for 20 min. The complex was added according to the manufacturer's protocol. The above sequences used for siRNA cell transfection are listed in Fig. β-catenin promoter, and pRL-TK plasmid. After 30 h, the Dual-Luciferase Reporter Assay System (Promega) was used to test relative luciferase and Renilla activities according to the manufacturer's instructions.HEK293T cells were transfected with a DNA mixture containing the SOX2 expression plasmid pLV-SOX2, pGL3-enhancer containing the 6) were cross-linked by 1% formaldehyde treatment. Glycine was used to stop the cross-linking. Chromatin was sonicated and sheared into small fragments following the instructions of the EZ-ChIP™ kit . One microgram of anti-RNA polymerase , 1 μg of IgG , or 1 μg of anti-SOX2 antibody  was added to pull down the target protein. Target protein-bound DNA was purified and subjected to PCR amplification of an ~200 bp fragment of the β-catenin promoter using the following primer sequences: FW: 5′-GCCGAGTGGAAACTTTTGTCG-3′, BW: 5′-GGCAGCGTGTACTTATCCTTCT-3′.Hypoxia-treated cells  were anaesthetized through intraperitoneal administration of sodium pentobarbital, and their hair was shaved with 70% ethanol. Full-thickness wounds were created on the middorsal skin with 8 mm disposable biopsy punches as described in our previous report . To confSkin tissues were excised, fixed in 4% paraformaldehyde, embedded and sectioned in paraffin, and stained with haematoxylin and eosin (H&E) . Digital images were taken by light microscopy .Immunohistochemical analysis was used to determine the expression of Snail and Slug. Formalin-fixed paraffin-wax-embedded skin biopsies were sectioned (4 mm) and incubated with antibodies against vimentin  and N-cadherin . The antibodies were detected using 3,3′-diaminobenzidine tetrahydrochloride to create a brown reaction product, counterstained with haematoxylin , and examined by light microscopy.t-test if the data satisfied the normality requirement. A two-tailed Wilcoxon-test was used to test variances between the two groups if the data did not satisfy the normality requirement. Statistical significance among three or more groups was assessed by one-way analysis of variance (ANOVA). Differences between two or more groups at different time points were estimated using two-way ANOVA. The Bonferroni method was used for multiple-group comparisons post-ANOVA. A P value of <0.05 was used to indicate statistical significance .All experiments were performed at least in triplicate to be eligible for the indicated statistical analysis. Statistical comparison was then performed with GraphPad Prism 5.0 (GraphPad Software Inc.). All data were presented as the mean ± SEM. The Shapiro-Wilk test was used to check whether the data were normally distributed. Differences in the results between two groups were analysed using a two-tailed unpaired 2) . IF analysis revealed that the expression levels of N-cadherin increased and those of E-cadherin decreased, as accompanied by a long and fibroblast-like shape, after 12 h of treatment with hypoxia  remarkably downregulated SOX2 protein levels in HaCaT cells Figures , and theβ-catenin gene), LEF1, and LGR5, which were enriched in the Wnt pathway according to KEGG pathway analysis, were first detected by qRT-PCR in hypoxia-mediated HaCaT cells  environment simply maintains ROS production at a low physiological level, avoiding uncontrolled ROS formation. Here, our results provide a concept for the existence of a mechanism underlying the maintenance of hypoxia-induced ROS at a balanced level in keratinocytes subjected to moderate hypoxic conditions (2% O2), which requires further investigation.Insufficient blood supply to wound tissue and heightened oxygen consumption lead to diminished oxygen availability. In fact, the O studies , 10. In xylation , 27, 28.xylation . ROS ovexylation . In thisIn addition to the role of ROS harmful factors, ongoing studies have revealed that ROS appear to be important signal mediators involved in physiological processes in the cell , 32. ConApart from the role of ROS in ensuring hypoxia-mediated EMT and migration, we further explored the mechanism by which ROS promote keratinocyte EMT and migration under hypoxia. According to the literature, low levels of intracellular ROS, as second messengers, induce various downstream signalling effects that contribute to the regulation of cell fate . Recent Although it is also well known that SOX2 plays a critical role in stabilizing the pluripotency of adult stem cells , there iβ-catenin was observed in keratinocytes under hypoxia. LEF1 is a β-catenin-responsive gene, and the stem marker Lgr5 is a target gene of β-catenin transcriptional activity [β-catenin expression [β-catenin in hypoxia-exposed keratinocytes. Through luciferase-based gene reporter and ChIP-PCR assays, β-catenin was demonstrated to be a potential molecular target of SOX2 in keratinocytes under hypoxic conditions. Utilizing selective inhibition, we further showed that keratinocyte EMT and migration under hypoxia are mediated by SOX2-induced Wnt/β-catenin activation. Based on the above results, we assumed that SOX2 exerted a stimulatory effect by activating the Wnt/β-catenin pathway in hypoxic keratinocytes. Meanwhile, the present study helps advance our knowledge of the role of the Wnt pathway in keratinocyte EMT, which is rarely reported and requires further investigation.The transcription factor SOX2 contributes to the regulation of gene transcription, thereby mediating cell fate as well as axis and pattern formation . Slug haactivity . Additiopression . Hence, ex vivo skin culture model in response to hypoxia was employed. Further results demonstrated the important role of ROS in the enhancement of SOX2 expression and Wnt/β-catenin pathway activation, thereby enhancing Slug expression in tissue. Topical application of low doses of ROS was demonstrated to enhance wound closure in mice [β-catenin are involved in ROS-associated modulation of reepithelialization of the wound area. In a further study, we will investigate whether the regulation of SOX2 can affect the activation of Wnt/β-catenin and thereby reepithelialization in wound healing.Finally, we explored the potential mechanism by which wound-induced ROS regulate skin repair. First, an  in mice . Here, w in mice . In addiβ-catenin in hypoxic keratinocytes (β-catenin pathway activation. These findings support strategies to target these relevant molecules and pathways for improving wound healing.In conclusion, our study documents for the first time the novel role of ROS in the determination of EMT and movement via the enhancement of SOX2 and activation of Wnt/inocytes . We furt


Unfortunately, the results on the microscopic scale are rarely applied to the preparation of macroscopic composite materials. Herein, the process of MXene functionalized CFs were attempted to be extended to short carbon fibers (SCFs) and used to strengthen epoxy materials. The results show that the cross-scale reinforcement of MXene functionalized SCFs can be firmly bonded to the epoxy matrix, which significantly improves the mechanical properties. Compared to neat epoxy, the tensile strength (141.2 ± 2.3 MPa), flexural strength (199.3 ± 8.9 MPa) and critical stress intensity factor  are increased by 100%, 67%, and 216%, respectively.The surface modification technology of carbon fibers (CFs) have achieved considerable development, and it has achieved great success in improving the interfacial shear strength (IFSS) of the polymer matrix. Among them, MXene (Ti In this way, the polar component and dispersion component of the CFs were increased at the same time, and the IFSS was increased by 186%.Carbon fibers (CFs) have a series of advantages, such as high specific strength, high specific modulus, high fatigue resistance, high corrosion resistance, and low linear coefficient of thermal expansion, so it is an ideal structural material for reinforcing polymer matrix ,2,3,4. In+1XnTx , M refers to early transition metals, and X refers to C or N element [x in the formula represents the surface terminations, such as O, OH, F, and/or Cl, which are bonded to the outer M layers [MXene is a transition metal carbide and nitride with a graphene-like structure 2D materials ,17,18. D element ,32,33,34M layers ,36,37,38M layers . TherefoAlthough the interface combination of modified CFs and polymer have been improved, it is still challenging to re-weave them into bundles or cloth for further applications. The CFs products pre-woven into the finished products are inaccessible to the modified substance due to the overlap between the fibers, which greatly reduces the modification effect. Therefore, short carbon fibers (SCFs) or surface-modified SCFs have attracted attention in the field of polymer reinforcement ,43,44,453C2Tx) to obtain the surface-modified cross-scale reinforcement. After finding out the most suitable load of ASCFs, the epoxy-matrix composites with the same amount of ASCFs functionalized by MXene were prepared. The results show that an appropriate amount of ASCFs can improve the mechanical properties of epoxy resin. On this basis, the ASCFs functionalized by MXene can further enhance the mechanical properties. The characterization results of the fracture indicate that the MXene functionalized ASCFs have a stronger bond with the epoxy matrix. Compared to the neat epoxy, the tensile strength, flexural strength and critical stress intensity factor (KIC) of composites can be increased to 100%, 67% and 216% at the maximum, which has a good application prospect.The present paper attempts to extend the successful surface modification method of continuous CFs to SCFs for epoxy reinforcement. ACFs were prepared into acid-treated SCFs (ASCFs) to prepare ASCFs/epoxy composites with different ASCFs contents. Simultaneously, ASCFs were functionalized by MXene  were cleaned with refluxed acetone  at 60 °C for 96 h to remove any polymer sizing and pollutants and yield untreated CFs. The untreated CFs were then oxidized in concentrated nitric acid  at 80 °C for 4 h. Subsequently, the carbon fibers were taken out and washed several times with de-ionized water  until pH approached 7. Next, the obtained fibers were dried in a vacuum furnace to yield ACFs. The ACFs were mashed and ground in an agate mortar, and passed through a 325-mesh sieve 3 times. The sieved powders were washed with de-ionized water, then centrifuged at 3500 rpm for 5 min. After the precipitate was thoroughly dried, the ASCF with a clean surface and an average length of ~20 μm were obtained. Finally, the ASCF was immersed in a solution with a concentration of MXene of 1 mg mL−1 for 15 min, washed with de-ionized water after being taken out and dried in a vacuum oven to obtain MXene functionalized ASCFs, as shown in Comprehensive details regarding all of the materials and the preparation of Tir papers ,46,47. ADGEBA  and MTHPA  were mixed in a mass ratio of 100:85. After the mixture was softened at 80 °C, ASCFs were added and ultrasonicated for 1 h. The 0.3 wt.% 2,4,6-Tris (dimethylaminomethyl) phenol  was added after the ultrasonic treatment, and then it was cured in a vacuum drying oven at 90 °C for 1 h and 110 °C for 4 h. According to the different addition amounts of ASCFs, epoxy-matrix composites with mass fractions of 1%, 2%, 3% and 4% were prepared and denoted as C1/epoxy, C2/epoxy, C3/epoxy and C4/epoxy, respectively. According to the results of tensile and flexural tests, the cross-scale reinforcements contain 2 wt.% ASCFs were added to DGEBA/MTHPA to prepare the MXene-functionalized ASCF/epoxy composite and denoted as C2M/epoxy.−1. The storage modulus and loss factor  of neat epoxy and composites were carried out under a nitrogen atmosphere from 25 to 790 °C at a heating rate of 10 °C min−1. Rectangular specimens (60.0 mm long × 8.0 mm wide × 3.0 mm thick) were used for flexural testing and loaded with a span of 48 mm at a crosshead speed of 2.0 mm min−1. The elastic modulus was calculated by the ratio of the stress difference (σ2–σ1) to the corresponding strain difference (ε2–ε1), i.e., ε2 = 0.25% and ε1 = 0.05%. Similarly, the flexural modulus was also calculated by the ratio of the stress difference (σfM2–σfM1) to the corresponding strain difference (εfM2–εfM1), i.e., εfM2 = 0.25% and εfM1 = 0.05%. At least five specimens were tested under each set of conditions. The flexural strength (σfM) and flexural strain (εfM) was calculated by using Equation (1) [F is the maximum load of the load-displacement curve for bending specimens, L is the span, b is the width of the sample, s is the deflection, h is the thickness of the sample.The tensile and flexural tests were performed at room temperature using an Instron 3367 mechanical testing machine  following ISO 527-1:1993 and ISO-178-2010, respectively. Standard dumbbell-shaped specimens (75.0 mm long × 12.5 mm wide × 2.0 mm thick) with a length of the narrow region of 25 mm were prepared for tensile testing at a rate of 1.0 mm min−1 to achieve sufficient loading time before the end of each test. The critical stress intensity factor  specimens (60.0 mm long × 8.0 mm wide × 3.0 mm thick). A sharp notch was machined at the midpoint of each specimen (4 mm in depth), and a natural pre-crack was generated by tapping a new razor blade into the notch. SENB specimens were also tested by Instron 3367 using a three-point-bending rig. Owing to the brittle nature of epoxy, the test speed was set to 0.5 mm mintion (3)  as follo and (5)  as folloGIC) was calculated as Equation (6) [E is the elastic modulus of the MXene/epoxy composites, ν denotes Poisson’s ratio of DGEBA/MTHPA system, and the value of 0.29 was used [The critical energy release rate (tion (6) :(6)GIC=Kwas used .−1) was determined when using MXene to functionalize ASCFs, so MXene does not accumulate too much on ASCFs at this concentration, as shown in 3C2Tx layers.The SEM images in TGA was utilized to investigate the thermal stability of each composite under a nitrogen atmosphere . All TGAStorage modulus is an index reflecting the elastic properties and influenced by the interfacial interactions between the filler and resin matrix of polymer composites . As showg). Compared to the neat epoxy, the Tg values of the composite decrease with the addition of reinforcement. Polymer composites have a large relaxation temperature range [g of the former is slightly lower, which further proves the influence of solid interface on the cross-linked network. The storage modulus value at room temperature and Tg are shown in re range ,56,57,58SEM was used to compare the fracture behavior of ASCF/epoxy composites and MXene functionalized ASCF/epoxy composites after the tensile test . The ten1/2), which is 9% higher than C2/epoxy (2.14 MPa·m1/2), and is significantly 216% higher than neat epoxy (0.74 MPa·m1/2). The propagation of crack is severely hindered through the fillers, which leads to the dissipation of higher fracture energy in composites. Further, the critical energy release rate (GIC) of C2M/epoxy has increased dramatically by ~650% than neat epoxy.Fracture toughness of ASCF/epoxy composites with different filler loadings and C2M/epoxy composites were evaluated by the SENB method, as shown in As shown in 3C2Tx concentration of 1 mg mL−1. ASCF/epoxy composites with low-loads in 1 wt.%, 2 wt.%, 3 wt.% and 4 wt.% were prepared, and the mechanical properties of the composites are the best at a load of 2 wt.%. For comparison, the cross-scale reinforcements with ASCFs’ content of 2 wt.% were also composite with epoxy resin. The experimental results show that ASCFs functionalized by MXene form a stronger interface interaction with the epoxy matrix, which effectively improves the mechanical properties of the epoxy composite. In general, continuous CFs surface modification technology has been successfully applied to SCFs reinforced epoxy, providing a technical route with high-performance application prospects.ASCFs with an average length of ~20 μm were prepared by grinding, sieving, washing and centrifuging nitric acid-treated CFs. A cross-scale enhancement of MXene functionalized ASCFs were prepared by immersing ASCFs in a solution with the Ti


HEK293 cells are one of the most widely used cell lines in research, and HEK293 cells are frequently used as an in vitro model for studying the WNT signaling pathway. The HEK293 cell line was originally established by transfection of human embryonic kidney cells with sheared adenovirus 5 DNA, and it is known that that HEK293 cells stably express the adenoviral E1A and E1B-55k proteins. Here, we show that HEK293 cells display an unexpected distribution of key components of the WNT/β-catenin signaling pathway where AXIN1, APC, DVL2 and tankyrase are all co-localized in large spherical cytoplasmic aggregates. The cytoplasmic aggregates are enclosed by a narrow layer of the adenoviral E1B-55k protein. The reduction of E1B-55k protein levels leads to the disappearance of the cytoplasmic aggregates thus corroborating an essential role of the E1B-55k protein in mediating the formation of the aggregates. Furthermore, HEK293 cells with reduced E1B-55k protein levels display reduced levels of transcriptional activation of WNT/β-catenin signaling upon stimulation by the Wnt3A agonist. The demonstrated influence of the E1B-55k protein on the cellular localization of WNT/β-catenin signaling components and on transcriptional regulation of WNT/β-catenin signaling asks for caution in the interpretation of data derived from the HEK293 cell line. The WNT/β-catenin signaling pathway plays a pivotal role during development, organogenesis, stem cell maintenance and self-renewal of many tissues, and aberrant WNT/β-catenin signaling activity is associated with the development of several disease conditions including cancer ,2. The mCell lines are important tools in cell biology research, and one of the most commonly used cell lines is human embryonic kidney (HEK) 293 cells ,11. The Given the broad use of HEK293 cells in WNT signaling research, we characterized the subcellular localization of key components of the WNT/β-catenin signaling pathway. We show that while β-catenin is predominantly localized at the cell membrane, AXIN1, APC, DVL2 and TNKS all co-localize in large cytoplasmic aggregates that are enclosed by a narrow layer of the adenoviral E1B-55k protein present in HEK293 cells. We further analyzed E1B-55k protein interaction partners and identified the AMER1 protein as a potential link between WNT signaling components and the E1B-55k protein in HEK293 cells. Finally, we demonstrated that siRNA-mediated reduction of E1B-55k protein levels lead to a dispersal of the cytoplasmic bodies and that HEK293 cells with reduced E1B-55k protein levels display reduced WNT/β-catenin transcriptional activation upon stimulation with Wnt3A.2.The human embryonic kidney cell lines HEK293 (ATCC CRL-1573) and HEK293T (ATCC CRL-3216) were obtained from the American Type Culture Collection . The stable reporter HEK293-STF/Ren cell line was described previously [To stimulate or inhibit WNT signaling activity, cells were treated for 24 h with Wnt3A CM or G007-LK, respectively. Wnt3A CM was prepared by collecting the supernatant from L Wnt3A cells (ATCC CRL-2647) according to the manufactures protocol. Unless otherwise stated, HEK293 cells were treated with Wnt3A CM diluted 50% in complete DMEM. Stock solutions of G007-LK   were fixed in 4% paraformaldehyde (Merck) (15 min at room temperature (RT)) permeabilized with 0.1% Triton-X100 (Merck)/PBS (15 min at RT) followed by incubation with primary and secondary antibodies diluted in PBS with 4% bovine serum albumin (Merck) (1 h RT each). Nuclear counterstaining was performed with DAPI (Merck) , and the coverslips were mounted in ProLong Diamond Antifade Mountant (Thermo Fisher Scientific). The following primary antibodies and dilutions were used: β-catenin , Tankyrase-1/2 (H-350) , Tankyrase-1/2 (E10) , AXIN1 (C76H11) , APC (F-3) , DVL2 (10B5) , E1B-55k (2A6) , Total HEK293 cell lysates were prepared by lysing cells in ice-cold lysis buffer , and complete protease inhibitor cocktail (Merck) for 20 min on ice. For Co-IP, Dynabeads protein G (Thermo Fisher Scientific) was used according to the manufacturer’s instructions. In brief, antibodies  and (E1B-55k (2A6) )g for 10 min, trypsin was inactivated by adding 100 µL 1% TFA, and the samples were again centrifuged at 14,000× g for 10 min. The resulting peptides were desalted and concentrated before mass spectrometry by the STAGE-TIP method using a C18 resin disk (3M Empore). Each peptide mixture was analyzed by a nEASY-LC coupled to QExactive Plus  with EASY Spray PepMap®RSLC column (Thermo Fisher Scientific) . The resulting MS raw files were submitted for protein identification using Proteome Discoverer 2.1 (Thermo Fisher Scientific) and Mascot 2.5  search engines. The search criteria for Mascot searches were: trypsin digestion with two missed cleavage allowed, carbamidomethyl (C) as fixed modification and acetyl (N-term), Gln->pyro-Glu (N-term Q), and oxidation (M) as dynamic modifications. The parent mass tolerance was 10 ppm and MS/MS tolerance 0.1 Da. The SwissProt database was used for the database searches. All of the reported protein identifications were statistically significant (p < 0.05) in Mascot and filtered in Proteome Discoverer Software (Thermo Fisher Scientific) for at least medium confidence identifications.Beads from the Co-IP were dissolved in 20 µL 0.2% ProteaseMAX Surfactant, Trypsin Enhancer in 50 mm NH4HCO3 followed by protein reduction, alkylation and on-beads digestion with trypsin overnight in 37 °C. After digestion, the samples were centrifuged at 14,000× Cells were transfected with the indicated siRNA construct  using the Lipofectamine 2000 transfection reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. The transfection mixture was kept on the cells for 24 h followed by growth medium replacement and incubation for another 48 h before fixation for immunofluorescence or preparation of cell lysates for Western blot analysis. The following siRNA sequences were used : Control siRNA: endoribonuclease-prepared siRNA pool (esiRNA) that that target multiple locations in the EGFP mRNA sequence , E1B-55k siRNA (I): (GGA GCG AAG AAA CCC AUC UGA UU) and E1B-55k siRNA (II): (GGC CAG AUU GCA AGU ACA AGA UU). E1B-55k siRNA constructs I and II targets nucleotide positions 3–23 and 542–562 in the E1B-55k CDS, respectively (from HAdV5 GenBank accession no. AY339865.1: 2019–3509).Quantification of average number of spherical TNKS bodies per cell was performed by counting number of spherical TNKS bodies per cell per cell in 10 randomly acquired images from control and E1B-55k siRNA-treated cells (>100 cells counted per treatment).Total cell lysates were prepared by lysing cells ice cold RIPA buffer (Merck) supplemented with complete protease inhibitor cocktail for 20 min on ice. Protein concentrations were quantified by the Pierce BCA Protein Assay kit (Thermo Fisher Scientific). Lysates  were electrophoresed (NuPAGE Novex 3–8% tris-acetate protein gels (Thermo Fisher Scientific)) and transferred to PVDF membranes (Merck). Following blocking of the PVDF membranes  in TBST, 1 h room temperature), immunodetection of proteins was performed by incubation with primary antibodies overnight at 4 °C and incubation with secondary antibodies for 1 h at room temperature. The following primary antibodies and dilutions were used: Tankyrase-1/2 (E10) , AXIN1 (C76H11) , DVL22 (30D2) , E1B-55k (2A6) Analysis of the effect of the E1B-55k protein on WNT signaling was performed using the HEK293-STF/Ren cell line where the β-catenin responsive TCF/LEF driven reporter (7xTCF/LEF binding sites regulating Firefly luciferase expression) and the constitutive CMV Renilla luciferase constructs were stably integrated . One dayt-test or one-way ANOVA as indicated in the figure legends.Testing for statistical significant difference between means was performed using the two-tailed unpaired To characterize the subcellular localization of central WNT/β-catenin signaling components, immunofluorescence (IF) staining was performed on untreated HEK293 cells and on cells incubated either with Wnt3A conditioned medium (CM) to activate the WNT signaling pathway or with the TNKS inhibitor G007-LK to inhibit WNT signaling activity . The subcellular localization and size of β-catenin degradosomes have been reported to vary between experimental systems and cell lines . HoweverTo investigate a possible role of the E1B-55k protein in the formation of the cytoplasmic aggregates and to gain insight into the protein organization in the TNKS/E1B-55k aggregates, siRNA was used to reduce E1B-55k protein levels, and high-resolution images were acquired by structured illumination microscopy (SIM) . SIM imaIn the E1B-55k, siRNA-treated cells as well as the cellular distribution of AXIN1, APC and DVL2 were changed from the accumulation in the E1B-55k aggregates to a uniform cytoplasmic distribution . These rTo identify E1B-55k interaction partners in HEK293 cells and to detect possible links between the E1B-55k protein and components of the WNT/β-catenin signaling pathway, co-immunoprecipitation (Co-IP) was carried out. Total HEK293 cell extracts were immune-precipitated (IP) with anti-E1B-55k antibodies and proteins present in the immunoprecipitates were identified by liquid chromatography-tandem mass spectrometry (LS-MS/MS) in two independent experiments . The lisGiven the observed E1B-55k-mediated accumulation of TNKS, AXIN1, APC and DVL2 in cytoplasmic aggregates , we nextIn this work, we showed that in HEK293 cells central components of the WNT/β-catenin signaling pathway display atypical cellular localizations by being sequestered in large cytoplasmic E1B-55k aggregates. By super-resolution microscopy, we established that these aggregates have a spherical structure that is surrounded by a narrow layer of E1B-55k protein. We found that reducing E1B-55k protein levels leads to the disappearance of the cytoplasmic aggregates, thus corroborating an essential role of the E1B-55k protein in mediating the formation of the aggregates. Lastly, our study reveals that HEK293 cells with reduced E1B-55k protein levels display diminished transcriptional activation of a WNT/β-catenin signaling reporter upon Wnt3A stimulation, thus demonstrating a functional effect of the aggregation on WNT signaling activity. The established impact of E1B-55k on both the cytoplasmic localization of key components of the WNT/β-catenin pathway and on the activity of WNT/β-catenin signaling in HEK293 cells asks for caution in the interpretation of data derived from this standard WNT signaling workhorse cell line.