Epoxy nanocomposites with float catalysis-produced CNT felt as a filler were prepared. Parameters such as the curing process, glass transition of epoxynanocomposites, structure and morphology of CNT felt, initial epoxy composition, and epoxy nanocomposites were investigated. The influence of CNT felt on curing process in epoxy nanocomposites with different amounts of curing agent was determined. An exothermic reaction between the curing agent and the surface of CNTs was established. It was found that the structure of epoxy nanocomposites has a high degree of heterogeneity: the presence of fiber-like structures and individualized CNTs is observed together with the regions that are typical for CNTs that are fabricated via a catalytic chemical vapor deposition (CVD). Based on the studies performed, it is possible to predict the production of epoxy nanocomposites with outstanding mechanical and thermophysical properties. In particular, the uncured compositions already obtained in this work can be used for the manufacture of electrically conductive glass and carbon fiber reinforced plastics and functional coatings. Carbon nanotubes (CNTs) in the form of a nonwoven fabric (felt) can be used as nanofillers of structural polymer composite materials (PCM) and to achieve a high degree of filling in PCM . One of CNT felt was used as a nanofiller of epoxy nanocomposites and was obtained by the method of float catalysis  on the p-preliminary obtaining a dispersion of CNT felt in acetone,-mixing the resulting dispersion with the epoxy composition ED 20 / DADFS,-drying the samples from acetone,-curing under the pressure according to the selected temperature program.ED 20 epoxy oligomer cured with 4,4′-diaminodiphenylsulfone (DADFS) was used as a binder for PCM. The PCM manufacturing process consisted of several stages:The dispersion of CNT felt in acetone was prepared by alternately using the Sonopuls-HD3400 submersible ultrasonic dispersant (exposure time 1 min) and IKAT 18 basic high-speed dispersant . Four cycles of exposure were used with breaks between cycles of 20 min for cooling during preliminary obtaining the dispersion of CNTs in acetone. The epoxy composition ED20/DDS was added to the resulting dispersion. Acetone was evaporated by holding the samples in an open container for 48 h at the room temperature and subsequent heat treatment in a vacuum box at a temperature of 60 °C for 2 h. The curing mode of the samples was chosen by the study of a gel formation using the method of dynamic mechanical analysis (DMA). The samples were pressed according to the regime: 140 °C for 1 h, 180 °C for 3 h. The thickness of the samples was set by the gaskets between the plates of the press.Thus, there were four types of samples prepared. The features and the differences of the samples are presented in MEASUREMENT PROCEDURESThe morphology and structural characteristics of the CNT felt were investigated by JEOL JSM-7600F scanning electron microscope (SEM) including thermal field emission with an accelerating voltage of 15 kV and resolution up to 1 nm, as well as by JEOL JEM-2010 transmission electron microscope (TEM). The content of residual catalyst in the felt, as well as the temperature of the onset of oxidation were determined by NETZSCH STA 449 F1 Jupiter (DTA/TGA) in the air flow of 70 mL/ min with a heating rate of 10 °C/ min from the room temperature to 800 °C. The specific surface area was determined by Autosorb-1c instrument and Ultrapyc 1200e gas pycnometer (Quantachrome Instruments). Temperature characteristics and changes in the enthalpy of various thermal processes of the initial epoxy composition and epoxy nanocomposites were obtained by differential scanning heat flow calorimeter (DSC) manufactured by MettlerToledo (Switzerland) in the air without the use of purge gas from the room temperature to 330 °C with a heating rate of 10 °C/ min. The temperature of the onset of the heat process on the DSC curve was determined as the point of intersection of the tangents to the peak and the baseline extrapolated to the inside of the peak. Changes in enthalpy  of various thermal processes were determined as the area above/ below the DSC curve. The glass transition temperature and the elastic modulus of the cured samples of the initial composition and epoxy nanocomposites were determined using a combination of dynamic mechanical analysis (DMA) and thermomechanical analysis (TMA) by thermomechanical analyzer by Mettler Toledo (Switzerland). The measurements were made in the air without using purge gas with a heating rate of 5 °C/ min from the room temperature to 240 °C. The measurement mode (type of deformation) was three-point bending. Free bending length was 10 mm. The dynamic load was from 0.05 N to 0.1 N with the frequency of 1 Hz. The glass transition temperature of the initial composition and epoxy nanocomposites were determined by TMA thermogram as the temperature of the onset of deformation caused by the transition of the polymer matrix from the glassy state to highly elastic state. The value of this temperature was determined as the intersection of the tangent to the initial linear part of the TMA curve with the tangent at the point of maximum strain rate.The curing mode of epoxy nanocomposites was chosen on the basis of a study of the gelation process by DMA. The measurements were carried out on a thermomechanical analyzer by Mettler Toledo (Switzerland) in the air without the use of purge gas in isothermal conditions at 140 °C. The preheating rate from the room temperature to 140 °C was 3 °C/min. The measurement mode (type of deformation) was compression. A sample of the composition with CNTs was placed between two layers of quartz fabric, then between two layers of thin aluminum foil. The resulting construction was placed in a cup of dense aluminum foil between two quartz disks. The upper disk was located under the indenter dynamically loaded with a force of 0.1N to 0.3N with a frequency of 1 Hz. The gelation time was determined by the characteristic inflection on the experimental time dependence of the elastic modulus at DMA curve. The specific conductivity of the cured nanocomposites was determined by the van der Pauw method. Samples of epoxy nanocomposites were studied by TESCAN VEGA 3 XMU electron microscope in the secondary (SE) electron mode at magnifications from ×100 to ×40,000 according to MI 1.2.042-2011. The fracture of the samples was obtained after treatment with liquid nitrogen, then they were glued with conductive glue to the microscope holder. The surface of the samples was subjected to ion-plasma etching on a JFC-1100 (JEOL) installation followed by sputtering of gold ≤ 10 nm in a Q150R ES magnetron sputtering system (Quorum Technologies). The study of CNT “felt” by electron microscopy showed that the structure of the main product is dominated by two-, three- and multi-walled nanotubes with a characteristic diameter of 2–40 nm. The tubes are mainly bundled .The high-temperature oxidation of CNT felt occurs in one stage , the iro2/g.Sorption studies have shown that the specific surface area is about 200 mThe DSC curves of the curing, the temperature of the maximum heat, and the magnitude of the thermal effect for the epoxy nanocomposites and the original composition are shown in It can be seen , Table 1An increase in the amount of curing agent in the system  leads to an acceleration of the curing reaction. The temperature of the max of thermal effect shifts to low temperatures by 7–10 °C; the thermal effects of curing are compared for the initial composition and epoxy nanocomposites. At the temperatures far beyond the range recommended for the use of cured composition  the destruction processes start showing themselves as peaks around 310 °C.The DSC curves of the cured samples ENC-1 (stoich) and ENC-2 are presented in Moreover, according to thermogravimetric analysis in the air, the introduction of CNTs into the epoxy composition did not change the nature of the high-temperature oxidation of ENC-1 (stoich) in the region of the onset of mass loss compared to the initial epoxy composition. At the same time, a small increase in the temperature of the onset of oxidation was noted. It can be assumed that the filler plays a role of a protective agent that slows down the processes of high temperature oxidation. It leads to an increase in heat resistance compared to the initial polymer .The DSC curves of nanofillers without epoxy oligomer that were used to prepare ENC-1 and ENC-2 and DSC melting curve of DDS are shown in As can be seen from After heat treatment of CNTs with DDS according to the regime mentioned before, neither the low-temperature endothermic peak, nor the first exothermic peak is observed, which suggests that DDS was immobilized at the surface of CNTS and thus became inactive in curing. As can be seen from the presented results, the elastic modulus of epoxy nanocomposites at the room temperature is 20–40% higher than the elastic modulus of the initial epoxy composition . The glaOne of the possible reasons for such a small change in the elastic modulus of the epoxy nanocomposite is a high degree of heterogeneity of their microstructure .As can be seen from the above photographs, the ENC-1 contains fiber-like structures b. CNTs oBoth ENC-1 and ENC-2 have fragments that are typical for CNTs fabricated by CVD pyrolysis (square frame). Therefore, it can be suggested that the structure of CNT felt includes CNT fragments that are subject to destruction at lower stress than intergrowths of “long” CNTs.Despite the high concentration of CNTs, the uncured ENC-2 can probably be used for the manufacture of electrically conductive glass and carbon plastics, as well as functional coatings. The specific conductivity of the cured ENC-2 is 4 S/cm.Epoxy nanocomposites with float catalysis-produced CNT felt filler were prepared and studied. It was established that the presence of CNT felt decelerates the curing reaction and reduces its thermal effect compared to the initial epoxy composition in the case of a stoichiometric ratio of the components in epoxy nanocomposite. CNT felt accelerates the curing reaction in the case of an excess of curing agent, the thermal effect of such reaction is compared with the thermal effect of curing for the initial epoxy composition. It was shown that the curing agent can enter an exothermic reaction with the surface of CNTs. It leads to the individualization of CNTs in the epoxy nanocomposite and the formation of a polymer “coat” on them. It was found that the structure of the epoxy nanocomposites has a high degree of heterogeneity: the presence of fiber-like structures and individualized CNTs is observed together with the regions that are typical for epoxy nanocomposites with CNTs that are fabricated by CVD. Based on the studies performed, it is possible to predict the production of epoxy nanocomposites with outstanding mechanical and thermophysical properties. In particular, the uncured compositions already obtained in this work can be used for the manufacture of electrically conductive glass and carbon fiber reinforced plastics and functional coatings.


Oral submucous fibrosis (OSF) is a potentially malignant disease of the oral cavity. New molecular predictors are needed to identify the high risk of malignant transformation in potentially malignant oral lesions. Our purpose is to explore PTPRZ1 and p120/β-catenin in clinical tissues was detected. Then, PTPRZ1, p120, β-catenin, RhoA, Rac1, CDC42, cyclin D1, and c-myc expressions were detected by qRT-PCR and western blot. CCK-8 was applied to measure hOMF cells viability. Wound healing and transwell assay were applied to measure cell migration and invasion. Western blot and IF detected the distribution of p-p120 and p-β-catenin. Tumor formation experiment explored PTPRZ1 effects on OSF.  The expression of PTPRZ1, p120, and β-catenin were abnormally expressed in cancer tissues. PTPRZ1 regulated the phosphorylation of p120/β-catenin. Western blot and IF showed that in the oe-NC group, p-p120 and p-β-catenin were expressed in the cell membrane. p-p120 and p-β-catenin were expressed in the cytoplasm and nucleus of the oe-PTPRZ1 group. In vitro experimental results revealed overexpression of PTPRZ1 and β-catenin, and silencing of p120 promoted cell proliferation, migration, and invasion. The tumor volume and weight in the sh-PTPRZ1 group were significantly reduced. IHC revealed the positive rate of PTPRZ1 was also low.  PTPRZ1, p120, and β-catenin to promote OSF malignancy. Overexpression of PTPRZ1 regulated the phosphorylation of p120/ Oral submucous fibrosis (OSF) is a chronic, insidious, and progressive oral mucosal disease , 2. It iβ-catenin is an important pathway that regulates tumorigenesis. PTPRZ1 can enhance β-catenin protein expression in the nucleus of human renal cell carcinoma and participates in regulating proliferation by activating β-catenin and its downstream genes [Receptor-type tyrosine-protein phosphatase zeta (PTPRZ1) is a member of the RPTP family . PTPRZ1 am genes .β-catenin or γ-catenin in the E-cadherin complex and plays a vital role in stabilizing the E-cadherin-catenin complex [β-Catenin is a protein containing a central armadillo (ARM) domain (residues 140–664), amino-terminus domain (NTD), and carboxyl terminus domain (CTD) [β-catenin is related to metastatic oral squamous cell carcinoma (OSCC) with positive cervical lymph nodes [β-catenin.Catenin cell-cell adhesion complex plays a vital role in the development and progression of cancer . p120-ca complex . The exp complex . In addi complex . β-Catenin (CTD) . It is ein (CTD) , 20. Abnph nodes . Howeverp120/β-catenin axis might play a role in the malignant transformation of OSF. We verified that OSF malignant transformation is related to the PTPRZ1/p120/β-catenin axis in human tissues. In addition, we used arecoline-induced cell models to elucidate the mechanism of PTPRZ1 regulated the phosphorylation of p120/β-catenin to promote OSF malignant transformation and to clarify its value as a potential therapeutic target, so as to provide novel ideas and theoretical evidence for the prevention and treatment of OSF malignant transformation.Therefore, we hypothesized that the PTPRZ1/Forty participants were recruited from Xiangya Hospital of Central South University. Human normal oral mucosal tissue, early OSF tissue, middle OSF tissue, and OSCC tissue were collected. The groups were normal group, early-OSF group, medium-OSF group, and the OSCC group. We have obtained the written informed consent of the subjects before the start of the study and obtained the approval of the Medical Ethics Committee of Xiangya Hospital (No. 201703635).2. They were divided into the control (HNOEC cells), OSCC (CAL27 cells), OSF (hOMF cells), OSF + arecoline (hOMF cells), oe-NC (hOMF cells), and oe-PTPRZ1 (hOMF cells) groups. The OSF + arecoline group was treated with 0.2 μg/mL arecoline for 24 h in hOMF cells [β-catenin, the β-catenin sequence was linked to the vector LV003. According to the instructions, β-catenin-vector plasmid was transfected into hOMF cells using lipofectamine 3000 reagent. They were divided into the oe-NC group and the oe-β-catenin group.Normal oral epithelial cells (HNOEC cells), OSCC cells (CAL27 cells), and human oral mucosa fibroblasts (hOMF cells) were purchased from the Cell Bank of the Chinese Academy of Sciences. HNOEC cells were cultured in a Minimum Essential Medium containing 10% FBS (Thermo Fisher Scientific). CAL27 cells were cultured in DMEM containing 10% FBS and 1% penicillin/streptomycin . hOMF cells were cultured in 90% H-DMEM and 10% FBS containing 100 U/mL P/S medium. They were placed at 37°C, cultivated in a humidity chamber with 5% COβ-catenin, RhoA, Rac1, CDC42, cyclin D1, and c-myc expression levels. To put it simply, total RNA was extracted by the Trizol method, RNA was reversely transcribed into cDNAs in accordance with the instruction of a reverse transcription kit . SYBR Green qPCR mix (Invitrogen) was performed to test genes relative expression in ABI 7900 system. The relative level of the gene was calculated by the 2–ΔΔCt method with GAPDH as the internal gene. The primer sequence used in this study is shown in QRT-PCR was performed to detect PTPRZ1, p120, β-catenin , p-β-catenin , RhoA , Racl , CDC42 , cyclin D1 , C-myc  primary antibody, and β-actin  were incubated for 90 min at room temperature. At room temperature, TBST was washed three times, and the second antibody HRP goat anti-mouse IgG  and HRP goat anti-rabbit IgG  were incubated. After ECL exposure, Odyssey Infrared Imaging System  was applied to detect the protein band, and the internal reference was β-actin. Each group was tested three times.RIPA lysis buffer  was applied to extract the total protein from cells and tissues according to the instructions. The protein of each group was quantified according to the BCA protein determination kit, and the SDS-PAGE loading buffer  was mixed; the mixture was heated for 5 minutes in boiling water at 100°C. The protein was adsorbed on the PVDF membrane by gel electrophoresis, and then it was sealed at 2 hours with a 5% skim milk solution at room temperature. PTPRZ1 , p120 , p-p120 , β-catenin was detected by IHC in human and mouse tumor tissues. The slices were roasted at 60°C for 12 h; then the slices were dewaxed to water and heated to repair the antigen. A total of 1% periodic acid was added, and the endogenous enzyme was inactivated for 10 minutes at room temperature. PBS was washed 3 times for 3 minutes. PTPRZ1, p-p120 , and p-β-catenin  primary antibodies were incubated overnight at 4°C, and PBS was washed 3 times for 5 minutes. The secondary antibody was incubated at 37°C for 30 min. PBS was washed 3 times for 5 minutes. We dropped the prepared color developing agent DAB working solution of 50–100 μL, incubated for 5–10 min at room temperature, washed with distilled water, and blued them with PBS. All levels of alcohol (60–100%) were dehydrated for 5 min. After removal, it was placed in xylene for 10 min and then sealed with neutral gum and observed under a microscope. Each group was tested three times.The expression of PTPRZ1, p-p120, and β-catenin. The slides were removed and washed with PBS 2–3 times. Then slides were fixed with 4% paraformaldehyde for 30 min and PBS was washed 3 times for 5 minutes. Then, it was permeabilized with 0.5% TritonX-100 at 37°C for 30 min. After washing with PBS, 5% BSA was sealed at 37°C for 1 h, p-p120  and p-β-catenin  were incubated overnight at 4°C. PBS was washed 3 times for 5 minutes. Diluted CoraLite594-conjugated Goat Anti-Rabbit IgG  was added. Then they were incubated at 37°C for 90 min, and PBS was washed 3 times for 5 minutes. Then they were dyed with DAPI (Wellbio) at 37°C for 10 min. The plates were sealed and observed under a fluorescence microscope. Each group was tested three times.IF detected the distribution of p-p120 and p-4 cells. Then, the cells were cultured in a 37°C, 5% CO2 incubator for 12 h, 24 h, 48 h, 72 h, 96 h, and 120 h, respectively. After experimental treatment, 10 μL CCK-8 reagent was added in an incubator for 2 h. The microplate reader  was performed to measure absorbance at 450 nm to assess cell viability in each group. Each group was tested three times.The cells in the logarithmic growth phase were seeded in 96-well plates, and each well was seeded with 1 × 10When the cells grew at a fusion rate of 90%, the cells were washed once with sterile PBS, scratched with a pipette tip, and washed once with PBS. Then we removed the scratched cells and added serum-free DMEM high-sugar medium. Photographs were taken at 0 h and 24 h to detect cell migration. Each group was tested three times.6/mL, added 100 μL to the upper chamber of the transwell chamber and 600 μL of complete medium to the lower chamber. After incubating at 37°C for 24 h, the cells on the surface of the upper chamber were wiped with wet cotton swabs, fixed with 4% paraformaldehyde for 20 min, stained with 0.5% crystal violet for 5–10 min, and observed and photographed under a microscope. Each group was tested three times.The cells were collected using the transwell system, resuspended with serum-free DMEM, and inoculated into transwell plate lumen. The inner side of the chamber on the transwell plate was precoated with Matrigel basement membrane matrix (BD Biocoat). The Matrigel glue was dissolved overnight at 4°C and then diluted with precooled basal medium (Matrigel: medium) at the ratio of 1:3. 40 µL of Matrigel glue was added to precooled transwell chamber and incubated at 37°C for 2 h to make Matrigel glue solidify. Cells were counted with basal medium and adjusted to 1 × 106 cells/mL. A total of 150 μL propidium iodide (PI) was added and stained at 4°C for 30 min in dark. PI was excited by a 488 nm argon-ion laser and received by a 630 nm pass filter. Ten thousand cells were collected through FSC/SSC scatter plot. The gating technique was used to eliminate adhesion cells and fragments, and the percentage of each cell cycle on the fluorescence histogram of PI was analyzed. Each group was tested three times.The cell suspension was collected and centrifuged to obtain cell precipitate. PBS was washed 2–3 times to prepare single-cell suspension, and cells number was adjusted to 1 × 105 cells were collected. We added 500 μL of binding buffer to suspend cells. After adding 5 μL Annexin V-FITC  and mixing well, 5 μL propidium iodide (PI) was added. The reaction time was 15 min at room temperature and kept away from light. Flow cytometry  was performed within 1 h. Each group was tested three times.The treated cells were digested and collected with trypsin without EDTA. Cells were washed with PBS twice and centrifuged at 2,000 rpm for 5 min each time; about 5 × 10μm EDU culture medium. The cells were incubated overnight in 100 μL 50 μM EDU medium, and PBS was washed 2 times for 5 minutes. Then 50 μL cell fixation solution  was added, and it was incubated for 30 min at room temperature. After adding 50 μL 2 mg/mL glycine and incubating in a decolorizing shaker for 5 min, PBS was washed for 5 minutes. Next, 100 μL penetrant was added to decolorize, and it was incubated in a decolorizing shaker for 10 minutes. PBS was washed once for 5 minutes. We added 100 μL of 1× Apollo® staining reaction solution, and it was incubated for 30 minutes in a decolorizing shaker at room temperature and kept away from light. Then we added 100 μL of penetrant to decolorize and washed 2–3 times on decolorizing shaker for 10 minutes each time. After adding 100 μL methanol to wash 1–2 times for 5 minutes each time and washing once with PBS for 5 minutes, DNA was stained and observed immediately after staining. Each group was tested three times.According to the EDU detection kit (RiboBio), the proliferation rate of cells was detected. First, the EDU was marked. The EDU solution was diluted with cell medium at the ratio of 1,000:1 to prepare an appropriate amount of 50 Twelve SPF-grade, 4-week-old mice were randomly divided into OSCC and sh-PTPRZ1 group with 6 mice in each group. Animal studies were approved by the Medical Ethics Committee of Xiangya Hospital (No. 20173637). The short hairpin targeting PTPRZ1 (sh-PTPRZ1) synthesized by Sangon Biotech and corresponding negative control sh-NC were used to knock down PTPRZ1 expression. The OSCC group was injected with sh-NC treated OSCC cells, and the sh-PTPRZ1 group was injected with sh-PTPRZ1 treated OSCC cells, and they were injected into nude mice axilla . The tumβ-catenin. One-way ANOVA was used for comparison between the two groups. p < 0.05 was considered statistically significant.Graphpad 8.0 was applied for statistical analysis, and experimental data were expressed as mean ± SD, which was repeated at least 3 times. Pearson correlation coefficient analyzed the correlation between PTPRZ1 and p120, PTPRZ1 and β-catenin in OSF tissues and OSCC tissues, qRT-PCR was first performed. The results revealed the expression of PTPRZ1 and β-catenin in early-OSF, middle-OSF, and OSCC groups increased compared with the normal group, while the expression of p120 decreased  were increased compared with the control group (HNOEC cells). Compared with the OSF group (hOMF cells), PTPRZ1 mRNA and protein expressions were increased in the OSF + arecoline group , while the expressions of p-β-catenin and p120 were decreased. Compared with the oe-NC group (hOMF cells), PTPRZ1, p-p120, and β-catenin mRNA and protein levels were increased, and p-β-catenin and p120 expressions were decreased in the oe-PTPRZ1 (hOMF cells). At the same time, the content of p-P120 in the cytoplasm was higher than that in the nucleus, but the change trend in both was consistent. IF showed that p-p120 and p-β-catenin were expressed in the cell membrane in the oe-NC group (hOMF cells). In the oe-PTPRZ1 (hOMF cells), p-p120 and p-β-catenin were expressed in cytoplasm and nucleus  compared with the oe-NC group  increased compared with the oe-NC group (hOMF cells) after 12 h, 24 h, 48 h, 72 h, 96 h, and 120 h. As shown in Figures β-catenin increased in the oe-β-catenin group, while p-β-catenin expression decreased. Compared with the oe-NC group (hOMF cells), the cell proliferation in oe-β-catenin group increased; the S phase increased; the G0/G1 phase decreased; and the apoptosis decreased  mediated transcription [β-catenin on the plasma membrane [β-catenin, thereby preventing the accumulation of β-catenin nuclei and the excessive activation of the WNT pathway during EMT [β-catenin was mainly expressed on the cell membrane, and PTPRZ1 and P–P120 were detected in all parts of cells with the same change trend. We found that PTPRZ1 could affect OSF carcinogenesis by regulating the phosphorylation of p120/β-catenin.Studies have shown that chronic oxidative stress leads to the amplification and overexpression of PTPRZ1 protein tyrosine phosphatase, thereby activating the  pathway . In the et genes . PTPRZ1 cription . p120 wacription . p120 comembrane . p120 string EMT . We founβ-catenin tyrosine phosphorylation led to a loss of intercellular adhesion, which in turn led to elevated cytoplasmic β-catenin levels, migrated to nucleus, and bound to transcription factor TCF/LEF. These led to increased MYC and cyclin D1 expressions, stimulated cell cycle, and proliferation [β-catenin and inhibit cell proliferation, as well as reduce target genes cyclin D1 and c-myc expressions in renal cancer [β-catenin. P120 and its phosphorylation of Tyr228 could inhibit the proliferation and invasion of colon adenocarcinoma cells [β-catenin but also can downregulate Rac1 activity and inhibit cell invasion. On the contrary, overexpression of p120 isoforms 3A could cause CDC42 inactivation and RhoA activation and has a small effect on invasion [β-catenin can also promote cell proliferation, migration, and invasion. In nude mice tumor formation experiments, it was found that silencing PTPRZ1 can inhibit the development of OSCC. These showed that PTPRZ1/p120/β-catenin was related to the development process of OSF.It was reported that feration . PTPRZ1 l cancer . Our resma cells . Previouinvasion . It has β-catenin phosphorylation to promote the malignant transformation of OSF. Our research provided ideas for the pathogenesis of OSF carcinogenesis. It was clear that the PTPRZ1/p120/β-catenin axis might serve as a potential therapeutic target and will help enrich new therapeutic strategies for OSF.Our results showed that overexpression of PTPRZ1 could regulate p120/


Hepatocellular carcinoma (HCC) ranks the 4th leading cause of cancer mortality in the world . AccumulTCGA-LIHC) data. Upon integrative analysis, we showed enrichment of the interstrand cross-link repair pathway, in which ubiquitin conjugating enzyme E2 T (UBE2T) was found to be most upregulated [via regulation of cell cycle related genes [To explore for critical molecules/pathways involved in the regulation of liver CSCs, we have performed an integrative analysis of three publicly available datasets involving stemness-related data (GSE5975 and CSCdb) , 5 and Hegulated . UBE2T ied genes .via ubiquitination [via ubiquitin-mediated degradation at K48. It remains a question whether UBE2T ubiquitinates Mule directly instead of acting as a mediator to transfer ubiquitin to E3 ligases. The direct action of E2 enzymes in the regulation of substrates via ubiquitination is not impossible. This piece of exciting data may suggest a new noncanonical role of UBE2T in cancer. Having said that, we cannot exclude the possibility that UBE2T binds to Mule and promote Mule autoubiquitination and degradation. Therefore, further in-depth investigation is required to elucidate the exact mechanism of how UBE2T degrades Mule.UBE2T was previously reported to regulate protein expression of BRCA1 tination . Based otination . Specifivia degradation of Mule in HCC cells. Firstly, we found a positive correlation between UBE2T and β-catenin protein expression upon alterations of UBE2T expression in HCC cells. Furthermore, UBE2T overexpression augments β-catenin transactivating activity, as evidenced by nuclear accumulation of β-catenin protein, increase in TOP/FOP activity and expression of β-catenin downstream genes. To further confirm that β-catenin is the major effector of UBE2T-mediated CSC functions, we have treated UBE2T repressed HCC cells with CHIR99021, a GSK3β inhibitor. CHIR99021 rescued the suppressive effects of UBE2T inhibition in UBE2T-repressed HCC cells. This in vitro data is echoed by the clinical observation that UBE2T was positively correlated with β-catenin in a cohort of HCC samples. Collectively, these results reveal that UBE2T may regulate liver CSC function, at least in part through the β-catenin signaling cascade.Based on the observation that β-catenin was directly degraded by mule , we furtIn summary, our study provides a novel prescriptive to understand the physiological and functional roles of UBE2T in cancer. Moreover, our study opens a new therapeutic avenue for targeting HCC by the selective blockade of UBE2T/Mule/β-catenin signaling cascade , warrant


Liver cancer is a dreadful tumor which has gradually increased in incidence all around the world. One major driver of liver cancer is the Wnt–β-catenin pathway which is active in a subset of these tumors. While this pathway is normally important in liver development, regeneration and homeostasis, it’s excessive activation due to mutations, is detrimental and leads to tumor cell growth, making it an important therapeutic target. There are also some unique characteristics of this pathway activation in liver cancer. It makes the tumor addicted to specific amino acids and in turn to mTOR signaling, which can be treated by certain existing therapies. In addition, activation of the Wnt–β-catenin in liver cancer appears to alter the immune cell landscape making it less likely to respond to the new immuno-oncology treatments. Thus, Wnt–β-catenin active tumors may need to be treated differently than non-Wnt–β-catenin active tumors.CTNNB1) or loss-of-function mutations in AXIN1 , are seen in a major subset of HCC. Because of the important role of β-catenin in liver pathobiology, its role in HCC has been extensively investigated. In fact, CTNNB1 mutations have been shown to have a trunk role. β-Catenin has been shown to play an important role in regulating tumor cell proliferation and survival and in tumor angiogenesis, due to a host of target genes regulated by the β-catenin transactivation of its transcriptional factor TCF. Proof-of-concept preclinical studies have shown β-catenin to be a highly relevant therapeutic target in CTNNB1-mutated HCCs. More recently, studies have revealed a unique role of β-catenin activation in regulating both tumor metabolism as well as the tumor immune microenvironment. Both these roles have notable implications for the development of novel therapies for HCC. Thus, β-catenin has a pertinent role in driving HCC development and maintenance of this tumor-type, and could be a highly relevant therapeutic target in a subset of HCC cases.Hepatocellular cancer (HCC), the most common primary liver tumor, has been gradually growing in incidence globally. The whole-genome and whole-exome sequencing of HCC has led to an improved understanding of the molecular drivers of this tumor type. Activation of the Wnt signaling pathway, mostly due to stabilizing missense mutations in its downstream effector β-catenin (encoded by  The protein later termed Wnt1 was first identified almost 40 years ago in the context of its proto-oncogenic nature . SubsequAPC) gene product, and diversin, also includes two kinases, casein kinase 1 (CK1), and glycogen synthase kinase 3 (GSK3), which sequentially phosphorylate β-catenin, priming it for recognition by the ubiquitin ligase β-TrCP [In the canonical pathway, the control of the Wnt-dependent cellular processes is achieved by a tight regulation of the amount of β-catenin—a transcriptional co-activator and a regulator of cell–cell adhesion. Normally, in the absence of Wnt signals, cytosolic levels of β-catenin remain low due to continuous proteasomal degradation of the protein, initiated by its destruction complex. The complex, composed of the scaffold Axin, tumor-suppressor adenomatous polyposis coli  in the liver . Wnt–β-CIntriguingly, a recent study also showed an important role of the Wnt–β-catenin pathway in serving a dual role of not only inducing hepatocyte proliferation but also maintaining hepatocyte function during liver regeneration after surgical resection, as well as after acetaminophen-induced injury and repair. Using single-cell RNA-sequencing, Walesky et al. showed a clever “division of labor” by the hepatocytes in the remnant liver following surgery or toxicant injury . This stAnother unique characteristic of the liver is the expression of unique genes by the hepatocytes based on their location within a microscopic hepatic lobule. This disparate gene expression allows for the hepatocytes to perform distinct functions that are necessary for the delivery of optimal hepatic output in terms of metabolism, synthesis, and detoxification, which are the broad categories of around 500 functions that hepatocytes perform to maintain health and homeostasis. Toward this end, Wnt–β-catenin signaling is known to be the major regulator of the expression of genes in the zone-3 or pericentral region of the metabolic lobule ,35,36. TThus, broadly, β-catenin seems to be playing a role in hepatocyte proliferation in physiological states including hepatic development  and liver regeneration , as well as in regulating hepatocyte functions including basally in the hepatocytes contained in zone-3 of the metabolic lobule. It is pertinent to mention the existence of regulators of the Wnt–β-catenin signaling that have been shown to play a role in the aforementioned hepatic processes. Factors like R-spondins and their receptors LGR4/5 have been shown to potentiate the effects of the Wnt–β-catenin pathways and have been specifically shown to positively impact the processes of both liver regeneration and metabolic zonation ,41.In addition to β-catenin being the major effector of Wnt signaling, it plays another evolutionarily conserved role at the adherens junctions (AJs), where it links the cytoplasmic tail of E-cadherin to α-catenin and F-actin . Since tThe regulation of β-catenin at the AJs in the hepatocytes is not completely understood. There is an incomplete understanding of whether the same pool of β-catenin is allocated to Wnt signaling and AJs, of when and how this allocation occurs, and of how dynamic this process is . The β-cAnother important observation made in the livers of mice lacking β-catenin in hepatocytes was the maintenance of intact AJs. This coincided with an increase in γ-catenin or plakoglobin, a normal inhabitant of the desmosomes. Indeed, in the β-catenin knockouts, γ-catenin was shown to co-precipitate and thus bind to E-cadherin in lieu of β-catenin ,51. ThisThe incidence of hepatocellular carcinoma (HCC) has risen steadily in the US and worldwide over last decades ,56. AnalMost HCCs are a consequence of years of hepatic damage and wound healing. The events leading up to HCC are complex and involve bouts of cell injury and death, immune cell infiltration, oxidative stress, and stellate cell activation . The livCTNNB1 mutations [It is important to emphasize the key phosphorylation sites located in exon-3 of β-catenin, which are important in its eventual degradation. When the Wnt signals are absent, β-catenin is sequentially phosphorylated at serine-45 (S45), S33, S37, and threonine-41 (T41) by casein kinase I (CKI) and glycogen synthase kinase 3β (GSK3β) . Phosphoutations ,63. Thesutations . Some hiAXIN1 are another major contributor to HCC development. AXIN1 is also among the top five mutated genes in HCC, seen in around 8% of human HCCs. This gene normally encodes for a protein essential for β-catenin degradation. In the absence of a functional Axin-1, β-catenin levels are increased and Wnt signaling is activated. Indeed, in preclinical models which used sleeping beauty transposon/transposase to express shRNA-Axin1 along with Met proto-oncogene in either a hepatic β-catenin-sufficient or deficient liver, the requirement of β-catenin was unequivocally shown in this model [Loss-of-function mutations in is model . IntriguTERT promoter or TP53 [Analysis of early HCC, multinodular HCC, and comparison of primary and metastatic HCC has also indicated that β-catenin has a trunk role in HCC similar to other major drivers such as mutations in  or TP53 .CTNNB1 mutations significantly correlate with the presence of other mutations such as in TERT promoter, NFE2L2, MLL2, ARID2, and APOB [CTNNB1 mutations also are seen to co-occur with the overexpression/activation of Met, Myc, or Nrf2 [CTNNB1 mutations and Met overexpression/activation, and their co-expression in murine liver in the Met–β-catenin model leads to clinically relevant HCC [The hepatic overexpression of β-catenin or the expression of mutated, constitutively-active β-catenin alone is insufficient for HCC development, as reported in many mouse models, suggesting cooperation with other pathways ,66. Indeand APOB ,67. CTNN or Nrf2 ,68,69. U or Nrf2 . For exavant HCC ,71. LikeThe five-year survival of liver tumors is 19.6%, attributable partially to lack of effective therapies . For locCTNNB1 knockdown resulted in a marked decrease in the viability and proliferation of human hepatoma cells in vitro [Ctnnb1-mutation-driven HCC, β-catenin inhibition using locked nucleic acid antisense (LNA) had a profound impact on tumor development [CTNNB1. This led to a notable decrease in tumor burden, also demonstrating β-catenin to be a highly relevant target in HCC for cases driven by CTNNB1 mutations.Because β-catenin is active in a notable subset of HCCs, and is also considered a trunk mutation, its inhibition could have a major impact on the treatment of a subset of these tumors. Several proof-of-concept studies in HCC, both in vitro and in vivo, have demonstrated the relevance of inhibiting β-catenin as a treatment strategy for HCC. siRNA-mediated in vitro . Similarin vitro . Interesin vitro . The Dsiin vitro . Using aelopment . More reCTNNB1 and the mutations in AXIN1. While there have been several other mechanisms identified to modulate β-catenin signaling, including the upregulation of certain Wnt genes, Frizzled genes, and epigenetic loss of negative regulators like DKK and FRPs and others, their true relevance remains unclear since Wnt–β-catenin signaling, like other signaling pathways, is able to regulate its overall activity via robust post-translational mechanisms. However, mutations in CTNNB1 or AXIN1 deem the β-catenin protein non-degradable and hence cannot be regulated by the normal mechanisms, which converge on β-catenin degradation to control the signaling pathway activity. This also suggests that several classes of Wnt inhibitors will not work in HCCs because they inhibit or impair Wnt activity upstream of the observed mutations in CTNNB1 or AXIN1. Hence, the suppression of β-catenin itself using the RNA-based therapies discussed in the preceding section, or those impairing β-catenin nuclear translocation, impairing its interaction with TCF4 or preventing the β-catenin–TCF complex from transactivating target genes, would be most effective in treatment of some subsets of HCC. Finally, identifying unique opportunities related to β-catenin signaling in HCC is important, as it may help in selecting or excluding the right group of patients, and may help to identify innovative opportunities to target other mechanisms that are intimately related to β-catenin activation unique to HCC.The most important mechanism of β-catenin activation in HCC are the mutations in Targeting β-catenin itself using RNA-based therapies is highly desirable. Several classes of siRNA- and antisense-based therapies have been described for use against β-catenin. The use of EnCore lipid nanoparticles along with Dicer substrate small interfering RNAs is especially innovative because it can be modified to specifically deliver the payload to liver tumors, and the safety of their use has been shown in patients . Others There may be an opportunity to identify the mechanisms of the nuclear transport or nuclear export of β-catenin. Targeting molecules that cargo β-catenin to the nucleus or activate its export out of the nucleus could have efficacy in the treatment of β-catenin-mutated HCCs. Pegylated interferon-α2a (peg-IFN), previously a first-line therapy for hepatitis C virus (HCV) patients, was shown to induce the levels of Ran-binding protein 3 (RanBP3), which is known to export β-catenin out of the nucleus . Peg-IFNCTNNB1 [The use of small-molecule inhibitors that interfere with its interactions with TCF or other relevant co-factors or components of the transcriptional complex would be highly desirable. However, a high specificity of the small-molecule inhibitors will be required because of the overlap of the β-catenin–TCF4 binding site, and with the binding sites for APC and E-cadherin . Even thCTNNB1 .In addition to a general role of β-catenin in regulating tumor cell proliferation, survival, and angiogenesis, there are specific and unique aspects of β-catenin activation due to mutations in HCC which can have notable biological and therapeutic implications—especially related to a step towards precision medicine.+ T cell inflamed environment and chemokines and interferon signature within the tumor [+ T cell priming and infiltration by acting on Batf3-lineage CD103+ dendritic cells (DCs) and decreasing CCL4 production by inducing the expression of transcription repressor ATF3 [+ T cell proliferation [CTNNB1-mutated HCCs are resistant to anti-PD-1 [ICIs have revolutionized the treatment of many tumors, including HCC as can be seen by the FDA approval of nivolumab and pembrolizumab as second-line therapy and of atezolizumab (anti PD-L1) plus bevacizumab (anti-VEGFA), as first line treatment for unresectable HCC . Howeverhe tumor . Intriguhe tumor . This issor ATF3 ; disruptsor ATF3 ; and incferation . HCCs wiferation ,95. We hnti-PD-1 , and henCTNNB1 may be one additional contributor of an immune-deficient tumor microenvironment which may in turn lead to resistance to ICIs.One additional relevant mechanism in the liver might be through a known interaction of β-catenin with NF-κB in the hepatocytes and liver tumor cells . This inCTNNB1-mutant liver tumors decreases tumor burden in many models [Glul, which encodes GS in hepatocytes in zone-3 of the hepatic lobule [CTNNB1 mutations are simultaneously positive for GS and p-mTOR-S2448 in preclinical models and patients (The suppression of β-catenin in y models ,82. We my models . The Wntc lobule , and leac lobule  (Figure c lobule . We idenc lobule  in zone-patients B. We als


SETD1A, a member of SET1/MLL family H3K4 methyltransferases, is involved in the tumorigenesis of numerous cancers. However, the biological role and mechanism of SETD1A in non-small cell lung cancer (NSCLC) remain to be elucidated.The expression of SETD1A, NEAT1, EZH2, and β-catenin in NSCLC tissues and cell lines was detected by qRT-PCR, immunohistochemistry and western blotting. The regulatory mechanisms were validated by chromatin immunoprecipitation, co-immunoprepitation and luciferase reporter assay. The self-renewal, cisplatin sensitivity and tumorigenesis of NSCLC cells were analyzed using sphere formation, CCK-8, colony formation assays and xenograft tumor models.SETD1A expression was significantly increased in NSCLC and its overexpression predicted a poor prognosis of patients with NSCLC. Functional experiments showed that SETD1A positively regulated cancer stem cell property and negatively regulated cisplatin sensitivity in NSCLC cells via the Wnt/β-catenin pathway. Next, we found that SETD1A positively regulated the Wnt/β-catenin pathway via interacting with and stabilizing β-catenin. The SET domain is dispensable for the interaction between SETD1A and β-catenin. Furthermore, we identified that SETD1A bound to the promoters of NEAT1 and EZH2 to activate gene transcription by inducing H3K4me3 enrichment. Rescue experiments showed that SETD1A promoted the Wnt/β-catenin pathway and exerted its oncogenic functions in NSCLC, at least, partly through NEAT1 and EZH2 upregulation. In addition, SETD1A was proven to be a direct target of the Wnt/β-catenin pathway, thus forming a positive feedback loop in NSCLC cells.SETD1A and Wnt/β-catenin pathway form a positive feedback loop and coordinately contribute to NSCLC progression.The online version contains supplementary material available at 10.1186/s13046-021-02119-x. Lung cancer, one of the most prevalent malignant tumors, is classified into two categories according to pathological characteristics, namely small cell lung cancer and non-small cell lung cancer (NSCLC) . SubstanHistone 3 Lysine 4(H3K4) methylation, including monomethylation, bimethylation and trimethylation (H3K4me1/2/3), is generally considered to be a transcriptional activation mark due to its capability to recruit transcription factors/coactivators . SETD1A Aberrant activation of the Wnt/β-catenin pathway is a critical event in the genesis of various cancers . Β-catenIn this study, we found that SETD1A regulates cancer stem cell property and cisplatin sensitivity in NSCLC cells via activating the Wnt/β-catenin pathway. Mechanistically, SETD1A activates the Wnt/β-catenin pathway via interacting with and stabilizing β-catenin. Furthermore, we identified two direct SETD1A target genes, NEAT1 and EZH2, through which SETD1A promotes Wnt/β-catenin pathway activity and malignant behaviors. Importantly, we demonstrated that SETD1A is a direct target of the Wnt/β-catenin pathway, thus forming a feedback loop to promote NSCLC progression.2 at 37 °C. Recombinant Wnt3a was purchased from R&D Systems .Lung cancer cell lines (A549 and PC9), HEK293T cell line and normal bronchial epithelial cell line (BEAS2B) were purchased from cell banks of Shanghai Institutes of Biological Sciences . A549, HEK293T and BEAS2B cell lines were cultured in DMEM  with 10% fetal bovine serum  and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin). PC9 cells were cultured in RPMI 1640 medium (HyClone) with 10% FBS and antibiotics. All the cells were maintained in a humidified atmosphere of 5% COThe full-length sequences of SETD1A, β-catenin, NEAT1 and EZH2 were inserted into the pcDNA3.1 vector  to construct the SETD1A, β-catenin, NEAT1 and EZH2-overexpressing plasmids, respectively. The NEAT1, EZH2 and SETD1A promoter fragments  were inserted into the pGL3-basic vector  to construct luciferase activity reporter plasmids  for the promoter activity assay, respectively. The TCF/LEF binding site mutated SETD1A promoter reporter plasmid (SETD1A-P-mut) and mutant β-catenin plasmid with K312E and K435E mutations (β-catenin-DM) were generated by site-specific mutagenesis based on the SETD1A-P-wt plasmid and wild type β-catenin plasmid, respectively. The TOP flash plasmid, FOP flash plasmid and pRL-TK vector were obtained from MiaolingBio . A series of plasmids of Flag-tagged SETD1A mutants were generated by YouBio .Β-catenin small interfering RNA (siRNA) and the corresponding negative control were synthesized by GenePharma . The transfection of plasmids and siRNA was performed using Lipo8000™ transfection reagent  according to the manufacturer’s instruction. The lentiviruses delivering short hairpin RNAs (shRNAs) targeting SETD1A and scrambled control sequences were obtained from GeneChem . The target sequences of shRNA and siRNA are listed in Additional file Clinical samples were obtained from the First Affiliated Hospital of Xi’an Jiaotong University. This study was approved by the Ethics Committee of the First Affiliated Hospital of Xi’an Jiaotong University. Each participant signed a written informed consent form. The clinical and pathological information of the patients are provided in Additional file https://tcga-data.nci.nih.gov/tcga/). The GSE71498 [The RNA sequencing data of lung adenocarcinoma  and lung squamous cell carcinoma  were downloaded from The Cancer Genome Atlas (TCGA) database  according to the manufacturer’s instructions. The extracted RNA was reverse transcribed using EasyQuick RT MasterMix . Realtime PCR was performed using 2 × RealStar Green Fast Mixture  on a CFX96 RealTime PCR system  according to the manufacturer’s instructions. The relative expression levels were calculated using the 2escribed . The priTotal protein was extracted using SDS lysis buffer (Beyotime). The nuclear and cytoplasmic protein was isolated using Nuclear Protein Extraction Kit  according to the manufacturer’s instruction. The protein was separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes . The membrane was blocked with 5% non-fat milk and incubated with primary antibodies at 4 °C overnight. Next, the membrane was washed 3 times and incubated with secondary antibodies for 90 min at 37 °C. The protein bands were visualized using an enhanced chemiluminescence kit (Millipore) on a ChemiDoc XRS+ imaging system . The primary antibodies used for western blotting were as follows: anti-SETD1A , anti-EZH2 , anti-CD133 , anti-OCT4 , anti-SOX2 , anti-β-catenin , anti-CCND1 , anti-MYC , anti-phospho-β-Catenin (Ser675) , anti-phospho-β-Catenin (Ser33/37/Thr41) , anti-Ubiquitin , anti-PKAα cat (A-2) , anti-APC (F-3) , mouse anti-Flag , rabbit anti-Flag , anti-HA , anti-GSK3α/β , anti-ICAT , anti-AXIN2 , anti-DKK1 , anti-GAPDH , anti-Vinculin  and anti-Lamin B1 .NSCLC cells were fixed with 95% ethyl alcohol at room temperature for 30 min and permeabilized with 0.2% Triton X-100 (Beyotime) for 15 min. After blocking with 5% BSA (Solarbio), the cells were incubated with the anti-Setd1A antibody , anti-β-catenin antibody  normal rabbit IgG  and normal mouse IgG  at 4 °C overnight. Next, the cells were incubated with Alexa Fluor 488-labeled Goat Anti-Rabbit IgG (H + L)  and Cy3-labeled Goat Anti-Mouse IgG (H + L)  for 1 h at room temperature. After that, the cells were stained with DAPI  to visualize the nuclei. Finally, the cells were viewed and imaged under a laser scanning confocal microscope .Immunohistochemical staining was conducted as previously described . The ant3) were seeded in 96-well plates overnight and subsequently incubated with the indicated cisplatin concentrations for 48 h. The final concentrations of cisplatin were 0 μM, 1 μM, 2 μM, 4 μM, 8 μM, 16 μM and 32 μM, respectively. Next, 10 μL of CCK-8 solution was added to each well and incubated for 2 h at 37 °C. The absorbance at a wavelength of 450 nm was measured using a microplate reader (Bio-Rad).The In vitro cisplatin sensitivity of NSCLC cells was determined by Cell Counting Kit-8  according to the manufacturer’s protocol. NSCLC cells  at a density of 1000 cells/well. The cells were cultured in DMEM/F-12 medium (HyClone) supplemented with 20 ng/mL epidermal growth factor , 10 ng/mL basic fibroblast growth factor , 5 μg/mL insulin (Invitrogen) and B-27™ Supplement (Invitrogen) for 14 days. Then the spheres larger than 50 μm in diameter were counted and photographed under a light microscope , 26.ChIP assay was performed using EpiQuik™ Chromatin Immunoprecipitation Kit  according to the manufacturer’s instructions. NSCLC cells were cross-linked and sonicated as previously described . The sheThe CoIP assay was performed using Protein A/G PLUS-Agarose  according to the manufacturer’s instructions. For two-step CoIP, the first IP was performed using anti-Flag M2 beads (Sigma-Aldrich). Then the complex was eluted with 3 × Flag peptide  and subjected to the second IP with anti-HA or control IgG. The protein samples from each step were analyzed by western blot analysis. The antibodies used for CoIP assay were anti-Setd1A antiboty  and anti-β-catenin antibody , mouse anti-Flag , anti-HA  and normal rabbit IgG .5) were plated in 24-well plates overnight and co-transfected with the indicated plasmid/siRNA or corresponding negative control and wild-type or mutant luciferase reporter vectors using Lipo8000™ Transfection Reagent. After transfection for 48 h, firefly and Renilla luciferase activities were measured using the Dual Luciferase Reporter Gene Assay Kit (Beyotime).A549 cells (1 × 106) stably transfected with shSETD1A or scramble lentivirus were suspended in 100 μL of serum-free RPMI-1640 medium and subcutaneously inoculated into the axillary of mice (n = 5). The tumor volumes were measured every 3 days and calculated with the formula: V = 1/2 × L × W2 . All the mice were sacrificed after 28 days. The tumors were resected and subjected to immunohistochemical staining and qRT-PCR analysis. For tumor initiating assay, 1 × 105, 1 × 104 or 1 × 103 SETD1A knockdown or negative control cells were subcutaneously injected into 4-week-old mice (n = 6). Tumor formation was monitored weekly for 8 weeks. The tumor initiating cells ratio or cancer stem cells frequency was calculated using the extreme limiting dilution analysis (http://bioinf.wehi.edu.au/software/elda/).Four-week-old male BALB/cA-nu/nu mice were purchased from Beijing Huafukang Biosciences . The animal experiments were approved by the Experimental Animal Committee of Xian Jiaotong University. For tumor propagation assay, PC-9 cells . Statistical To investigate the role of SETD1A in NSCLC, we first employed RNA sequencing data from TCGA database to validate the expression SETD1A in NSCLC. We found that SETD1A levels were increased in NSCLC tissues compared with those in the normal lung tissues Fig. : Fig. S1To investigate the function of SETD1A in NSCLC cells, we used two shRNAs to stably knock down SETD1A expression in NSCLC cell lines Fig. A-B. WestPrevious studies have proven that SETD1A promotes Wnt/β-catenin pathway activity in colorectal cancer and neural progenitor cells , 20. ThuTo explore the mechanism by which SETD1A regulates the Wnt/β-catenin pathway, we first examined the influence of SETD1A on β-catenin expression. We found that SETD1A positively regulated the total and cytoplasmic β-catenin protein levels, while the transcript levels were not significantly affected Fig. E. These The phosphorylation and subsequent ubiquitination of β-catenin plays an important role in β-catenin stability . Thus, wWe further investigated the domain of SETD1A responsible for binding to β-catenin. A series of plasmids of Flag-tagged SETD1A mutants and one HA-tagged β-catenin plasmid were used to perform immunoprecipitation assays in HEK293T cells Fig. A. The reTo further explore the mechanism by which SETD1A regulates the Wnt/β-catenin pathway, we analyzed the RNA sequencing data in the GSE71498 and GSE52230 datasets. We found that SETD1A knockdown in A549, MDA-MB-231 and HCT116 cells increased the expression of some Wnt/β-catenin negative regulators, such as AXIN2, ICAT, DKK1 and SIAH1 , we identified a putative TCF/LEF binding site (− 173 to − 167) within the TCF4-enriched region  and induced pluripotent stem cells (iPSCs), and gradually reduced during the differentiation of stem cells. Further investigation has indicated that SETD1A precisely modulates the pluripotency and self-renewal of stem cells in an H3K4me3-denpent manner , 21, 35.The Wnt/β-catenin pathway is a critical signaling pathway in maintaining cancer stem cell property as previously reported . In the Many mechanisms are involved in Wnt/β-catenin pathway activity regulation, including many Wnt pathway negative regulators. For example, SIAH1, GSK3β and AXIN2 all take part in the ubiquitination and subsequent proteasome-dependent degradation of β-catenin , 37, 38.Many studies have focused on the co-regulators and downstream target genes of SETD1A. However, they have rarely been involved in the upstream regulatory mechanism of SETD1A. We investigated whether canonical signaling pathways regulate SETD1A expression. By using ENCODE database, we identified a few pathways that might be involved in regulating SETD1A expression, including the Wnt/β-catenin pathway. We chose the Wnt/β-catenin pathway for the following experiments due to the possibility of forming a feedback loop with SETD1A. As shown in Fig. In summary, we found that SETD1A regulates cancer stem cell property and cisplatin sensitivity in NSCLC via activating the Wnt/β-catenin pathway. Moreover, SETD1A interacts with and stabilizes β-catenin to activate the Wnt/β-catenin pathway through the SET domain. Importantly, we identified that NEAT1 and EZH2 are two novel targets of SETD1A, through which SETD1A activates the Wnt/β-catenin pathway activity. In turn, β-catenin activates SETD1A transcription, thus forming a positive feedback loop to promote NSCLC progression. Based on this study, new prognostic factors and therapeutic targets may be determined, contributing to improvements in the diagnosis and prognosis of NSCLC patients.Additional file 1: Table S1. The oligonucleotides used in this study.Additional file 2: Table S2. The clinical and pathological information of patients included in this study.Additional file 3: Figure S1. Normal rabbits IgG staining of NSCLC specimens. Scale bar, upper 100 μm, lower 10 μm.Additional file 4: Figure S2. IC50 value for cisplatin in NSCLC cells as indicated was shown. Data are shown as the means ± SD. *P < 0.05, **P < 0.01Additional file 5: Figure S3. The effects of SETD1A on the Wnt/β-catenin pathway. A, Wnt/β-catenin pathway activity in A549 cells was detected by TOP/FOP flash reporter assay following SETD1A knockdown and overexpression. B, the nuclear β-catenin levels were analyzed by immunofluorescence assay following transfection with the empty vector and SETD1A plasmid. C, CTNNB1 transcript levels in NSCLC cells were detected by qRT-PCR following SETD1A overexpression. D, Cytoplasmic β-catenin levels was detected by western blotting following SETD1A knockdown and overexpression. E, The subcellular localization of SETD1A protein was predicted by the BUSCA webserver. F, The interaction between SETD1A and β-catenin in BEAS2B cells was analyzed by co-immunoprecipitation. G, β-catenin stability was analyzed by CHX chase assay following transfection with the empty vector and SETD1A plasmid. H, Immunofluorescence analysis using normal IgG in PC9 cells is shown. I, β-catenin expression was attenuated by MG132 treatment in SETD1A knockdown cells. J, PC9 cell lysates were immunoprecipitated using a β-catenin antibody and subjected to western blot analysis with the corresponding antibodies as indicated after Wnt3a treatment for 12 h. K, S675 phosphorylation of β-catenin was determined by western blotting following SETD1A overexpression without Wnt3a stimulation. Data are shown as means ± SD. *P < 0.05, **P < 0.01.Additional file 6: Figure S4. SETD1A promotes NSCLC progression via NEAT1/EZH2/β-catenin axis. A, Sphere formation ability in SETD1A knockdown cells was analyzed following transfection with the empty vector, β-catenin, NEAT1 and EZH2 expression vector, respectively. B, Cisplatin sensitivity in SETD1A knockdown cells was analyzed by colony formation following transfection with the empty vector, β-catenin, NEAT1 and EZH2 expression vector, respectively. The final concentration of cisplatin was 5 μM. C, Cisplatin sensitivity was detected by CCK-8 assay following transfection as indicated. Data are shown as means ± SD. *P < 0.05, **P < 0.01. (TIF 192 kb)Additional file 7: Figure S5. SETD1A knockdown increases the expression of Wnt/β-catenin pathway negative regulators. A, AXIN2, ICAT and SIAH1 expression in SETD1A knockdown and negative control group cells in GSE71498 dataset was analyzed. B, DKK1 expression in SETD1A knockdown and negative control group cells in GSE52230 dataset was analyzed. C, AXIN2, ICAT, SIAH1, DKK1 and GSK3β transcript levels in A549 cells were analyzed by qRT-PCR following SETD1A knockdown. Data are shown as means ± SD. *P < 0.05, **P < 0.01. D, DKK1 and AXIN2 protein levels in PC9 cells were analyzed by western blotting following SETD1A knockdown. E, ICAT and GSK3β protein levels in NSCLC cells were analyzed by western blotting following SETD1A knockdown. F, A positive correlation between SETD1A and NEAT1 expression in LUAD tissues was identified in StarBase online database. G, No correlation was identified between SETD1A and NEAT1 expression in StarBase online database. H-I, A positive correlation was identified between SETD1A and EZH2 expression in LUSC (H) and LUAD (I) tissues in StarBase online database.Additional file 8: Figure S6. H3K4me3 peaks in the NEAT1 promoter region in A549 cell line from ENCODE database were visualized in UCSC genome browser.Additional file 9: Figure S7. H3K4me3 peaks in the EZH2 promoter region in A549 cell line from ENCODE database were visualized in UCSC genome browser.Additional file 10: Figure S8. The relative enrichment of WDR5, H3K27ac and H3K27me3 in the NEAT1 and EZH2 promoters was detected by ChIP-qPCR assay. Data are shown as means ± SD. ns, not significant. *P < 0.05.Additional file 11: Figure S9. NEAT1 and EZH2 overexpression attenuates the effects of SETD1A knockdown on the Wnt/β-catenin pathway. A, NEAT1 expression in NSCLC cells transfected with the empty vector and NEAT1 expression vector was analyzed by qRT-PCR. B, EZH2 expression in NSCLC cells transfected with empty vector and EZH2 expression vector was analyzed by western blotting. C, ICAT and GSK3β expression in SETD1A knockdown cells was analyzed by western blotting following transfection with the empty vector, NEAT1 and EZH2 expression vector, respectively. D, Wnt/β-catenin pathway activity in SETD1A knockdown cells was analyzed by TOP/FOP flash reporter assay following transfection with the empty vector, NEAT1 and EZH2 expression vector, respectively. Data are shown as means ± SD. *P < 0.05, **P < 0.01Additional file 12: Figure S10. SETD1A is a target of Wnt signaling pathway. A, ChIP sequencing analysis of TCF7L2/TCF4 in cancer cell lines from the ENCODE database was visualized in UCSC genome browser. B, SETD1A expression was detected by western blot analysis after transfection with the empty vector, wild type β-catenin plasmid and mutant β-catenin plasmid (β-catenin-DM). β-catenin-DM, a mutant β-catenin plasmid with double mutagenesis of K312E and K435E.


Further, patients with high FBXW5 expression were found to have poor prognosis . Cell experiments revealed the promoting effects of FBXW5 on the proliferation, invasion, metastasis, and chemoresistance of GC cells. Blocking LATS1-YAP1 leads to the loss of FBXW5-mediated regulation of the Hippo pathway and partial functions. Further, co-immunoprecipitation and in vivo ubiquitination assays revealed the interaction between FBXW5 and LATS1, which promoted the ubiquitination and degradation of LATS1. Based on mouse xenograft assays, FBXW5 silencing attenuated the growth of subcutaneous tumor xenografts. Altogether, FBXW5 was found to inactivate the Hippo signaling pathway by enhancing LATS1 ubiquitination and degradation, which promoted the invasion, metastasis, and drug resistance of GC cells.F-box and WD repeat domain-containing 5 (FBXW5), with WD40 repeats, can bind to the PPxY sequence of the large tumor suppressor kinases 1/2 (LATS1/2) kinase domain, resulting in ubiquitination. Ubiquitination and the subsequent degradation of LATS1/2 abrogate the Hippo pathway and worsen gastric cancer (GC). However, the effects and molecular mechanisms of FBXW5 in GC remain unexplored. To elucidate the clinical significance of FBXW5, immunohistochemistry was conducted to reveal the positive correlation between FBXW5 expression and lymph node metastasis ( Approximately 670,000 new gastric cancer (GC) cases and 490,000 GC-related deaths are reported each year in China . PatientThe Hippo pathway is involved in GC cell malignancy –5. AbrogHomo sapiens spindle assembly abnormal protein 6 homolog (HsSAS-6), and promote its ubiquitination and degradation, thereby regulating centrosome duplication and affecting cell cycle regulation [FBXW5, a newly identified member of the F-box protein family, was initially found to inactivate interleukin (IL)-1β-induced JNK/p38 MAPKs and nuclear factor (NF)-κB, and the phosphorylation of Thr187 by transforming growth factor-β-activated kinase 1 (TAK1) . Thereafgulation . As a regulation . FBXW5 wgulation .Based on our results, a high level of FBXW5 underlies tumor invasion, lymph node metastasis, TNM stage, and poor prognosis in GC patients. FBXW5 inactivated the Hippo signaling pathway by binding to LATS1 and promoting LATS1 ubiquitination and degradation, which in turn promoted the invasion, metastasis, and chemoresistance of GC cells. Overall, the findings of this study provide novel insights into targeted therapy for GC.https://www.proteinatlas.org/) to determine the expression of FBXW5 in common human malignant tumors. FBXW5 mRNA was broadly detectable in common human malignant cancers, including GC, colorectal cancer, and lung cancer , was used to determine the significance of FBXW5 expression in GC. GC patients with high expression of the FBXW5 mRNA were found to exhibit poorer prognosis  than those with low FBXW5 expression , poor TNM stage (p = 0.018), and low differentiation (p = 0.042) (Table p = 0.02)  Fig. . A total02) Fig. , E. More02) Fig. .Table 1CFBXW5 was found to be expressed in multiple GC cell lines and was commonly higher in GC cells than in the GES-1 cell Fig. . FBXW5 wWe conducted CCK8 assays using gradient concentrations of 5-fluorouracil . The downregulation of FBXW5 increased the sensitivity of GC cells to 5-fluorouracil, whereas the upregulation of FBXW5 increased the resistance of cells to 5-fluorouracil Fig. . SimilarCTGF, CYR61, and c-Myc  and the Genotype-Tissue Expression (GTEx) databases with the webserver, GEPIA, FBXW5 was found to be positively correlated with the Hippo pathway target genes Myc Fig. . FurtherM1 Figs.  and S2B.M1 Figs. . In contM1 Figs. . HoweverM1 Figs. . ImmunofM1 Figs. .Fig. 4FBR = 0.44, p < 0.001)  Fig. . CollectChanges in the level of FBXW5 could lead to changes in the levels of the LATS1 and YAP1 proteins. To clarify the specific mechanism by which FBXW5 regulates the Hippo pathway, we performed co-immunoprecipitation (Co-IP) assays using HEK-293T cells and confirmed the binding of exogenous LATS1 protein with exogenous FBXW5 protein Fig. , B. FBXWWe validated the function of the FBXW5 protein in vivo by constructing a subcutaneous xenograft tumor model in nude mice. We transfected MGC-803 cells with lentivirus harboring shFBXW5 or scramble and generated a stable cell line through antibiotic selection. The silencing of FBXW5 was validated by western blotting, and subcutaneous inoculation was subsequently performed Fig. . The tumTo determine whether the mechanism underlying FBXW5 function is mediated by the LATS1-YAP1 axis, we silenced LATS1 in BGC-823 cells using siRNA. After LATS1 silencing, FBXW5 could not enhance the levels of YAP1 and its downstream molecule, CTGF Fig. , indicatTo determine whether the LATS1-YAP1-Hippo pathway makes an essential contribution to FBXW5-mediated functions, such as the regulation of EMT, malignant properties, and chemoresistance of GC cells, we knocked down LATS1 and FBXW5 in HGC-27 cells. When LATS1 was knocked down, the protein levels of N-cadherin, vimentin, and E-cadherin did not significantly change after FBXW5 silencing Fig. . The exoIn this study, clinical gastric tumor samples, cell-based experiments, and nude mouse models were employed to provide the first evidence that FBXW5 binds to and degrades LATS1 through the ubiquitin-proteasome pathway, ultimately inactivating the Hippo signaling pathway Fig. . These an = 79; validation cohort n = 120), patients with high FBXW5 protein expression were found to have a poorer prognosis than those with low FBXW5 protein expression.Proteins in the F-box protein family constitute a subunit of the SCF ubiquitin ligase complex and are crucial for phosphorylation-dependent ubiquitination . The F-bFailure to cure GC is primarily due to metastasis and chemoresistance. Our transwell and scratch assays revealed that FBXW5 contributes to GC cell invasion and migration. For EMT, epithelial cells were inclined to possess the mesenchymal cell phenotype. Herein, FBXW5 was found to positively regulate vimentin and N-cadherin expression and negatively regulate E-cadherin expression. Thus, FBXW5 may enhance GC cell invasion and metastasizing abilities by promoting EMT. Previously, FBXW5 was proposed to form a complex with CUL4A in non-small cell lung cancer, which led to the degradation of the tumor suppressor protein, DLC1, a Rho GTPase-activating protein, ultimately promoting cell proliferation . FBXW5 iCTGF, CYR61, c-Myc, and other downstream target genes of the Hippo pathway using qPCR. Co-IP assays revealed binding between FBXW5 and LATS1. However, no obvious binding was observed between FBXW5 and LATS2 (a homolog of LATS1) as well as the downstream effector, YAP1. FBXW5 was found to regulate the Hippo signaling pathway by binding to LATS1 and degrading LATS1 via ubiquitination. N-terminally located F-box motif and WD40 repeats domain were two recognized domains of FBXW family proteins, COOH-terminal WD40-repeat domain is often associated with substrate recognition. By constructing FBXW5 deletion mutants (ΔF-box and ΔWD), this study demonstrated that FBXW5 binds to LATS1 via the WD40 repeats suggesting that LATS1 may be the substrate of FBXW5 [CTGF, CYR61, c-Myc, and other oncogenes by FBXW5 was mediated via the LATS1-YAP1 axis. Finally, we delineated the significance of the FBXW5-LATS1-Hippo signaling pathway regulation using nude mouse models.To validate our hypothesis that FBXW5 regulates the Hippo pathway, we employed the bioinformatics analysis tool, GEPIA, and searched the databases, TCGA and GTEx. Subsequently, we silenced FBXW5 using a siRNA and confirmed the regulatory relationship between FBXW5 and of FBXW5 . The silβ-TRCP E3 ubiquitin-proteasome pathway [Recently, Mei Shi Yeo et al. reported that FBXW5 may promote GC tumorigenesis and metastasis by activating the FAK-Src signaling pathway, which indicated that FBXW5 might be a potential therapeutic target . Our stu pathway . Coincid pathway , 10. Acc pathway , 25. To Fresh patient tissues were retrieved from the General Surgery Department of First Affiliated Hospital of Nanchang University. All sixteen patients signed informed consent document before sample collection. A total of 79 paraffin-embedded patient tissues were collected between January 2010 and December 2010 for the training cohort, while 120 tissues were obtained between January 2011 and December 2012 for the validation cohort. Patients did not receive treatment before surgery. The study was approved by the Ethics Committee of the First Affiliated Hospital of Nanchang University.2 humidified incubator [The human immortalized gastric epithelial cell line, GES-1, the GC cell lines  and HEK-293T cells were obtained from the Shanghai Institute for Life Science, Chinese Academy of Sciences . Cells were cultured in RPMI-1640  containing 10% fetal bovine serum  at 37 °C in a 5% COncubator .The detailed IHC and immunofluorescence assay procedures are provided in our previous paper . The priThe specific sequences of siRNA are listed in Supplemental Table Western blotting and Co-IP analysis were conducted as previously described . MutatioThe protocols for total RNA extraction, reverse transcription and qPCR are provided in our previous paper . The priCell invasion and migration capacities were evaluated by transwell and wound healing assays, according to methods described previously , 27.Cell proliferation and colony formation assays were carried out to assess GC cell chemotherapy resistance. Transfected cells were treated with gradient concentrations of 5-fluorouracil or cisplatin. The detailed experimental procedures for the CCK8 and colony formation assays are described in our previous reports , 28.Ubiquitination assays were performed using HEK-293T cells, according to the detailed procedure provided in our published paper [A total of 12 female BALB/C nude mice (4 to 6 weeks old) were randomly divided into two groups (LV-shFBXW5 or LV-scramble) with six mice in each group. The animal protocol was approved by the Institutional Animal Care and Ethics Committee of the First Affiliated Hospital of Nanchang University. Scramble short hairpin RNAs or short hairpin RNAs targeting FBXW5 were subcloned into the lentiviral expression vector, GV248 (Genepharma). MGC-803 cells, lentivirus, and puromycin were used to construct FBXW5 stably silenced cells. The detailed animal experiment procedure is described in our previous report .t-test. Chi-squared tests were performed to assess the association between FBXW5 expression and clinicopathological characteristics. Survival curves were plotted by the Kaplan–Meier method using GraphPad Prism V8. SPSS Statistics 23  was used for all statistical analyses. A P value <0.05 was considered to indicate statistical significance (*P < 0.05 and **P < 0.01).Data are expressed as mean ± SEM of three independent experiments. The difference between groups was determined using unpaired two-tailed Student’s western blots original dataSupplementary figure and table legendsSupplementary TablesSupplementary Figure S1Supplementary Figure S2Supplementary Figure S3


Trichophyton rubrum (T. rubrum) is the main cause of chronic dermatophytosis which is highly prevalent worldwide. This study was aimed to fabricate and characterize polymeric emulgel of eugenol and linalool for the treatment of T. rubrum infections. Using the slow emulsification method, the emulgel was prepared and characterized for thermodynamic stability, pH analysis, viscosity, spreadability, swelling behavior, %drug content, surface morphology, globules size, polydispersity index, surface charge (mV), thermal behavior, in vitro drug release and XRD studies. Biological activities of emulgel were conducted against T. rubrum in vitro and in vivo. Results indicated that emulgel formulations were thermodynamically stable. The pH of the formulations was within an acceptable range for skin. The viscosity and spreadability were optimum for the better patient compliance. The swelling behavior was 111.10 ± 1.25% after 90 min. The drug content was within the official pharmacopeia limit i.e., 100 ± 10%. The surface morphology revealed by scanning electron microscopy showed a spherical-shaped structure with characteristic larger cracks and wrinkles. The droplet size, PDI, and surface charge of the optimized emulgel were 888.45 ± 8.78 nm, 0.44 and −20.30 mV, respectively. The emulgel released 84.32% of eugenol and 76.93% of linalool after 12 h. There was complete disappearance of the diffraction peaks corresponding to the drugs after XRD analysis. In rabbits, the infection was safely and completely recovered after 12 days and the emulgel produced significant effects (p < 0.05) similar to the standard product Clotrim®. It is concluded that the eugenol–linalool emulgel best described all its physical properties and can be applied topically for the treatment of T. rubrum infections. Conventional semisolid dosage forms used for the topical application of drugs have several disadvantages. They may be sticky and have thick consistency. The patient feels difficulty in application of such kinds of ointments, lotions, and creams because sometimes they are applied on the skin by rubbing which produces severe discomfort. The stability of these preparations is another issue. To avoid these problems, polymer-based emulgels are now frequently used in the pharmaceutical and cosmetic industries .Emulgel is a blend of polymer-based gel and emulsion which is beneficial in the sense that it is suitable for the delivery of hydrophobic and hydrophilic drugs across the skin into the blood stream, providing better release of the drugs . EmulgelCinnamomum tamala, cinnamon, ginger, clove, dill, and star anise [Eugenol is a slightly pale or colorless oily liquid which contains an aromatic ring and belongs to the allylbenzene class of the aromatic compounds ,3. It isar anise ,5. It isar anise .Artemesia vulgaris, Cananabis indica, Cannabis sativa, Cinnamomum tamala, and Humulus lupulus [Linalool is a terpene alcohol naturally occurring in different plant flowers and spices. It has been estimated that more than 200 plant species are containing linalool. Lamiaceae, Rutaceae, and Lauraceae are the most famous plant families producing linalool in abundance . Linaloo lupulus . Linaloo lupulus ,10.T. rubrum is a dermatophyte fungus belonging to the phylum Ascomycota and its favorite habitat is the outer-most dead layer of the skin, where it colonizes and causes severe infection. The surface of the colony while inside of the colony may appear yellow to brown and red in color. The growth of T. rubrum is quite slow, it appears as peg-shaped or tear drop-shaped, and its growth is inhibited by compounds containing nitrogen, sulfur, and phosphorus. The in vitro and in vivo studies have shown that its isolates have been reported to produce penicillin [nicillin ,10,11.T. rubrum are usually transmitted by the use of contaminated garments, towels, and linens. T. rubrum attacks favorably on glabrous skin which is hair free [T. rubrum [T. rubrum infection is usually observed in the upper skin layer; however, deeper skin and muscles may also get infected by this fungus. It attacks the skin and may cause folliculitis, and in chronic cases it may cause the formation of granuloma of the skin especially in the patients of HIV [Infections caused by air free . Common . rubrum . T. rubrs of HIV .T. rubrum totally depends upon the nature and severity of the infection. Miconazole, tolnaftate, clotrimazole and terbinafine are commonly available drugs for the treatment of T. rubrum infections. Topical preparations containing antifungal agents are usually used for the skin infections of T. rubrum however nails infections are not treated with the application of topical preparations due to the lack of absorption of drug from the surface of nail to nail plate [Treatment of the fungal infection caused by il plate .The use of polymers in drug delivery systems or pharmaceutical dosage forms is considered safer particularly when they are intended for topical treatment of various microbial infections . CarbomeT. rubrum infections.Thus, this study was aimed to fabricate and characterize poly (acrylic acid)-based pharmaceutical emulgel co-loaded with eugenol–linalool for the treatment of ® 1% cream (Clotrimazole) by Zafa Pharmaceutical Karachi Pakistan.Carbopol 934 , Ethanol , Eugenol, Linalool , PEG-400 , Liquid paraffin , Methanol , Methyl parabene, Propyl paraben , Span 20 , Triethanolamine  Tween 80 , Potato dextrose agar (PDA) , Distilled water and ClotrimA local breed of male rabbits weighing of 1–2 kg were used in this study. The rabbits were purchased from the local market of Lahore and were put into steel cages. A bed of saw dust was applied uniformly in the cages. They were acclimatized for one week before the experiments and were provided free access to food and water ad libitum.The basic methodology for the formulation of an emulgel includes the preparation of an emulsion and then addition of a polymer as a gelling agent to form the emulgel. Emulgel was fabricated by the method reported by Burki et al. 2020), slightly modified . The fir020, sligEmulgel formulations were physically examined for the evaluation of homogeneity, color, consistency, phase separations, and grittiness . The forThe viscosity of emulgel preparation was determined by using viscometer  at 25 °C. The readings were taken at specified time in triplicates and results were averaged.The spreading coefficient of the emulgel was determined using a specially modified apparatus in the laboratory by the drag and slip method. The apparatus was made of a wooden block with a mounted a pulley on its one edge, and a glass slide fitted on the wooden block. About 2 g of the emulgel was applied over the surface of glass slide mounted on the wooden block. Another glass slide with the same dimensions was taken and it was put over the fixed glass slide mounted on the wooded block in such a way that the emulgel was sandwiched between two glass slides. A 1 kg block was put on the glass the slide for 5 min to remove trapped air and to ensure uniform spreading of the emulgel. Then an 80 g weight was hung by attaching a thread to the top glass slide with the help of a hook. The thread was passed above the pulley and time was noted. The smaller the time taken by the slide to cover a distance of 7.5 cm on the fixed glass, the better the spreading coefficient of the preparation . The folS represents “Spreadability of formulation”.M represents the “Weight tied to upper slide of the apparatus.L represents the “Length of glass slides”.and T represents the “Time taken to separate the slides from one another.Swelling behavior of the emulgel was found by taking 1 g of the emulgel on aluminum foil (Porous). The foil containing emulgel was then put into a small beaker with 10 mL of 0.1 N NaOH solution. A small amount of sample from the aluminum foil was taken out from the beaker and dried at room temperature. It was then reweighed, and the swelling index was calculated by the following formula :(2)SI = SI indicates swelling index.W1 indicates original weight of emulgel.W2 indicates weight of swollen emulgel.Drug content of the preparation was determined by using spectrophotometric method. Briefly, 1 g of the emulgel was taken in a test tube and mixed with 9 mL ethanol, centrifuged, and then filtered. The absorbance was recorded both for eugenol and linalool using UV-spectrophotometer . A standard solution was prepared in the same solvent and the absorbance was noted . The druAnalysis of surface morphology and apparent shape of emulgel was performed via scanning electron microscopy as reported in literature with slight modification . The samThe globule size (droplet size), PDI, and zeta potential of the prepared emulgel was tested by using Zetasizer  according to a study by Eid et al. (2014) slightly modified . For thiThermal behavior of the ingredients and emulgel was recorded by DSC and TGA. Under a nitrogen environment, a DSC of the ingredients and formulations was performed. Heat stress was gradually applied from ambient to 400 °C at a rate of 10 °C/min. TGA was conducted in a temperature range of 0–800 °C.The in vitro release of eugenol and linalool from the emulgel was determined using Franz Diffusion Cell . This instrument consists of two compartments: a donor compartment and a receptor compartment. A skin or artificial membrane is usually fixed between these two compartments. The formulation is applied or loaded on the donor compartment and the receptor compartment is filled with buffer solution. The samples  are withdrawn at specified period of time from the receptor compartment, and, at the same time fresh buffer is added to the receptor compartment to maintain the sink condition. The samples are then analyzed using UV visible spectrophotometer. In our study, 1 g of emulgel was applied on the donor compartment by fixing a cellulose membrane. The receptor compartment was filled with phosphate buffer solution pH 7.2. The samples were withdrawn at 0 h, 1 h, 2 h, 4 h, 8 h, and 12 h, and then analyzed by UV visible spectrophotometer .The XRD studies of the ingredients and physical mixture, i.e., emulgel, were conducted by a Bruker AXS D-8  fitted with a CuK radiation detector. This was operated at a node voltage of 40 kV and the input current was 30 mA .The optimized emulgel was then tested for its antifungal activity. The media used for the growth of fungi was potato dextrose agar (PDA) which was spread in a thin layer over the glass plate. One hundred microliters of fungal suspension was taken and poured on an agar plate and spread uniformly with the help of a sterile swab. A sterile filter paper disc of about 6 mm containing 50 μL of emulgel was placed on the agar plates. These plates were then placed in an incubator at 37 ± 2 °C. After 72 h, the zone of inhibition and clearance were calculated and recorded in mm. The blank formulation was used as a control in all the experiments. All the values were calculated in triplicates.The current study was approved by the institution ethical review board of Gomal University, D.I.Khan Pakistan under reference no. 506/QEC/GU.n = 5). Group-I was the control group which did not receive any treatment, Group-II was standard group treated with commercial product Clotrim®  containing clotrimazole (Standard group), andGroup-III was test/experimental group treated with prepared emulgel of eugenol and linalool .The animals were divided into three groups containing five rabbits in each group (T. rubrum strain (ATCC) was provided by Microbiology Lab of University of Veterinary and Animal sciences Lahore Pakistan.2 area was abraded with sandpaper. About 0.1 mL suspension containing 1 × 107 cells of T. rubrum was applied to the marked area using a sterile pipette-tip and rubbed thoroughly.Dermatophytosis was induced by the method of Ghannoum et al., (2016) with slight modification . BrieflyThe treatments were repeated each day and continued for 12 days. The lesions were examined continuously from 1 day post-infection to 12 days post-infection . ClinicaThe SPSS  and EXCEL were used as statistical tool. Data were expressed as mean ± SEM. Student t test and one way ANOVA were applied to data set.In preparation of any emulgel, the physical properties of emulsion are mostly considered and the preparation which has high consistency, elegance, and stability is preferred . The resp > 0.05.Viscosity plays an important role in the delivery of drugs used via topical or transdermal application. Several characteristics of formulations, including the stability, spreadability, drug release, and ease of application, are dependent on the viscosity . The visThe extent to which a topically applied pharmaceutical formulation is spread on skin is termed as its spreadability . It is cThe action of the polymer in pharmaceutical preparation is enhanced when the macromolecule is fully swollen. The swelling provides rheology modification, suspending properties, and emulsification to the topical formulations . Polymerp > 0.05) between the percent drug content for both drugs. The results of the drug content revealed that %drug content was within the official limit  allowed by the United States Pharmacopeia (USP) [The uniform distribution of drugs in any pharmaceutical preparation is important for its therapeutic effect and can be confirmed by percent of drug content . The resia (USP) .The SEM analysis of the emulgel revealed an almost spherical-shaped structure with characteristic large wrinkles and cracks distributed throughout the surface A,B. The Droplet size has an important role in the formulations used for topical or transdermal delivery of drugs . SeveralThermal stability of all the individual ingredients and prepared emulgel was analyzed through thermo-gravimetric and DSC measurements, and results are presented in p > 0.05) between % release of drugs, i.e., eugenol and linalool from ELE2 formulation. The lower drug release from the formulation coukd be correlated to the presence of a gelling agent which makes a complex gel network and thus caused longer diffusion pathways of drug permeated through the membrane [The therapeutic efficacy of any drug depends upon the release of drug from pharmaceutical dosage forms . The relmembrane . The sammembrane .The XRD analysis of eugenol, linalool, and the physical mixture, i.e., emulgel was carried out to evaluate the solid-state transformation. The PXRD chart of eugenol and linalool displayed characteristics peaks at 11.7°, 15.7°, 17.2°, 21.4°, 22.5°, 23.6°, 30.2°, and 43.6°. These sharp and intense peaks demonstrate the crystalline nature of formulation. The distinctive peaks of the eugenol and linalool were not detected in the emulgel formulation. There was a complete disappearance of diffraction peaks corresponding to the eugenol and linalool portrayed by the PXRD patterns of drug-loaded emulgel. The results of the XRD analysis are presented in T. rubrum and the zone of inhibition was 5.5 mm. The blank formulation did not show any antifungal activity while eugenol–linalool-loaded emulgel (ELE2) formulation showed significant (p < 0.05) antifungal activity by inhibiting the zone (10.5 mm) of T. rubrum as compared to the pure eugenol and linalool. The detailed results are shown in T. rubrum was inhibited by eugenol and linalool. Linalool was more effective in growth inhibition of T. rubrum as compared to eugenol. It was also observed that co-loaded emulgel of eugenol and linalool significantly (p < 0.05) inhibited the growth of T. rubrum.The results of in vitro antifungal activity indicated that when eugenol and linalool were applied in the quantity of 50 μL, the growth of fungus was inhibited by 2.5 mm and 3.5 mm, respectively. Similarly, eugenol and linalool were used in combination against the T. rubrum growth.Thus, it can be concluded that the emulgel prepared by using a combination of linalool and eugenol is more effective in inhibition of In vivo studies were performed on rabbits and they were divided into three groups. First, infection was induced on the back of animals and the animals were observed for 72 h for the assessment of induction of infection. It was observed that maximum infection was induced in 72 h in all the groups treated with fungal strain. Details are shown in ® for 12 days, the infection was safely and completely recovered, and the relative score reached 0. It cured the infection in 10–12 days. Group-3 was treated with eugenol–linalool emulgel which cured the infection in 12 days. Thus, the emulgel produced results quite similar to those of the standard group. The fungal infections were recovered only if a large amount of active ingredients was released by the pharmaceutical dosage form that penetrates skin layers and was retained for a prolonged duration at the site of infection [In vivo antifungal activity was performed using rabbits as experimental animals after induction and confirmation of fungal infections. The results indicated that no recovery from infection was observed in Group-1 animals which were kept as a control group, and were given only a normal routine diet. The relative score of 5 was observed in Group-II (Standard group) animals which indicated that extensive damage to the skin and complete hair loss were observed in the rabbits. In the first three days, partial damage to the skin and hair loss was observed while in the next three days large areas of redness, scaling, exposed patches, and ulceration were observed, while in the next four days redness, swelling, bald patches, and scaly areas were seen. After treatment with marketed cream Clotrimnfection . Accordinfection . The detT. rubrum infections is highly stable and the physical properties of the emulgel remained feasible over an extended period of time. It is also concluded that the linalool and eugenol emulgel best described all its physicochemical features and can be safely applied topically to treat infections caused by T. rubrum.This study concludes that the poly (acrylic acid)-based pharmaceutical emulgel co-loaded with eugenol and linalool for the treatment of