Gardenia florida has been widely used for its antioxidative, hypoglycemic, and anti-inflammatory properties. However, whether G. florida fruit extract (GFFE) regulates hair growth has been rarely studied. This study was the first application of GFFE on hair growth both in vitro  and in vivo (C57BL/6 mice). The effects of GFFE on cell proliferation and hair growth-associated gene expression in hDPCs were examined. Moreover, GFFE was applied topically on the hair-shaved skin of male C57BL/6 mice, the hair length was measured, and the skin histological profile was investigated. GFFE promoted the proliferation of hDPCs and significantly stimulated hair growth-promoting genes, including vascular endothelial growth factor (VEGF) and Wnt/β-catenin signals, but suppressed the expression of the hair loss-related gene transforming growth factor-β1 (TGF-β). Furthermore, GFFE treatment resulted in a significant increase in the number, size, and depth of cultured hair follicles and stimulated the growth of hair with local effects in mice. In summary, the results provided the preclinical data to support the much potential use of the natural product GFFE as a promising agent for hair growth.As a herbal medicine, the extract from the fruits of  Recently, the number of hair loss patients has continued to increase, with a trend in younger people and females , 2. Hairβ-catenin signaling pathway [α-reductase [β [HDPCs play key roles in the proliferation, differentiation, and hair cycle control of hair follicles . Several pathway –20, pros pathway , and pro pathway  stimulateductase –26, TGF-ctase [β , FGF-5 [ctase [β , and proctase [β  are knowα-reductase and is not valid for women [Hair loss is recognized as a common dermatological problem, which significantly influences the patient quality of life and mental state, and the development of treatment agents has been markedly growing. Current drugs available for hair loss treatment include medications such as Minoxidil® and Finasteride® , 30. Minor women , 34.Therefore, the development of new ways to enhance hair growth and prevent hair loss is urgently needed in the hair care industry , 36. RecGardenia florida fruits have been used in antioxidative, anti-inflammatory, and hypoglycemic purposes [β1 and DKK1 decreased; however, the LEF1, Wnt5a, β-catenin, and VEGF increased; we also verified the effects of GFFE treatment on protein expression of TGF-β1, Wnt5a, and VEGF in hDPCs by western blot examination. Then, we investigated GFFE for its potential effect in the C57BL/6 mouse model showing the hair length difference, and histological findings verified that retardation of entry into catagen occurred in response to GFFE application. Taken together, these data considered that GFFE may be a new ingredient for hair growth-promoting and hair loss treatment potential.As a traditional herbal medicine that is widely distributed in most parts of China, purposes . This stG. florida fruit extract (GFFE) was purchased from the Nanjing DASF Biotechnology Co., Ltd . Human dermal papilla cells (hDPCs) were purchased from Sciencell Research Laboratories, Inc.2 at 37°C. Cultured cells were then treated with various concentrations of GFFE for 24 hours according to the kit protocol. CCK-8 solution was added, followed by a 4-hour incubation, and absorbance at 450 nm was measured on a TECAN Enzyme standard instrument Spark 10 M. The cell proliferation rates were calculated from OD and represented as percentages of negative control (NC).Cell proliferation was measured using Cell Counting Kit-8 . Human DP cells were seeded in 96-well plates at a density of 3,000 cells/well and incubated in a humidified atmosphere with air and 5% CO5) and incubated until grown to 80–90% confluence in a 10% FBS DMEM medium. After culture with serum-free DMEM medium for 24 h, the cells were added to each treatment and incubated for 24 h in aserum-free DMEM (NC), with 19.5 μg/mL of GFFE. Total RNA was extracted using Monzol™ Reagent  according to the manufacturer's instructions. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal reference. For RT-PCR, 1 µl each of the forward primer (10 ng/µl) and reverse primer (10 ng/µl) per well, iTaq universal Green supermix (2x) 10 µl, nuclease-free H2O 6 µl, and cDNA 2 µl were mixed to a final volume of 20 µl. The PCR procedure software recorded the average fluorescence value of each cycle of the reaction. The relative expression levels of different genes in the cells were obtained by comparing the Ct values. The experiment was repeated three times with three replicates seated per reaction.In vitro model, hDPCs were plated on a 6-well plate  with the following setting condition: 95°C for 10 min; 40 cycles including 95°C for 10 sec, 60°C for 30 sec, and 72°C for 30 sec. cDNA was amplified using MonScript™ RTIII All-in-One Mix with dsDNase .As for qRT-PCR, MonAmpTM ChemoHS qPCR Mix  was used following the instructions of the manufacturer. The qRT-PCR was performed using a BIO-RAD CFX96β1, 5′-GCAGAAGGATCACCACAACC-3′ (forward), TGF-β1, 5′-TGTCTGCACTGCGGAGGTAT-3′ (reverse) [β1-catenin, 5′-AACTTGCTCAGGACAAGGAA-3′ (forward), β1-catenin, 5′-TCCTAAAGGATGATTTACAGGTC-3′ (reverse) [Primers were used as follows: GAPDH, 5′-TGTTGCCATCAATGACCCCTT-3′ (forward), GAPDH, 5′-CTCCACGACGTACTCAGCG-3′ (reverse) ; VEGF, 5reverse) ; DKK1, 5reverse) ; β1-catereverse) ; LEF1, 5reverse) .β1  and rabbit anti-Tubulin . We used BeyoECL Star (Beyotime) reagent to detect protein.The cultured cells were collected and lysed in RIPA buffer (Beyotime) with protease inhibitors (Beyotime) after rinsing twice with PBS (precooled at 4°C). Protein was quantified with a BCA protein concentration determination kit (Beyotime), and then separated by SDS- polyacrylamide gel electrophoresis and electroblotted onto polyvinylidene fluoride membranes. After blocking in 5% nonfat milk for 2 h at 25°C, the membranes were probed with primary antibodies followed by horseradish peroxidase-conjugated secondary antibodies . Primary antibodies used were rabbit anti-VEGF protein , rabbit anti-Wnt5a , rabbit anti-TGF-n = 5) were used for this study. The dorsal area hairs (approximately 2 cm in width and 3 cm in length) on the back of the mouse in the telogen phase were shaved 24 h before applying the treatments. Animals in group 1 served as NC (water), and group 2 received 2% minoxidil as a positive control group. The animals in group 3, group 4, and group 5 received different doses  of GFFE, respectively. The 5 groups were applied topically to the test area with 0.2 mL once a day, for 30 days. The animals were isolated for an hour and then kept back in their respective cages. During the application period, differences between treatment and nontreatment groups in average body weight or abnormalities in the mice were not observed. The hair regrowth activities of GFFE were evaluated by observing and photographing the back skin of mice. On day 14, day 21, and day 30, 10 hairs were plucked from the mice's shaved area randomly from all groups and the average length of each treatment was measured.The animals were housed in a temperature-controlled room with a 12-h light/dark cycle and were allowed free access to food and water in the course of experiments. Twenty-five male five-week-old C57BL/6 mice  in 5 randomized groups (μm tissue section was stained with the standard hematoxylin and eosin (H&E staining) protocol [Dorsal skin tissues from each mouse were excised and fixed in 4% neutral formalin for 24 h. They were embedded in paraffin after routine tissue processing. For the hair follicle's histopathologic evaluation, a 4 protocol . HematoxOn 14, 21, and 30 days after beginning the animal experiment, 10 hairs were plucked from the mice's shaved area randomly from all groups and the average length of each treatment was measured.t-test for the comparison among groups using SPSS WIN (v25.0). The results were considered statistically significant if the p values were less than 0.05.All treatments were repeated at least three times. Data represent biological replicates. The data were statistically analyzed by Student's ∗), 110.67% (∗), and 112.92% (∗∗), respectively. Particularly, the 0.032% GFFE exhibited the highest cell proliferation rates than the NC group. Collectively, these results suggest that GFFE facilitates the proliferation of DPCs and improves the hair-inducing abilities of these cells.Dose-dependent proliferative effects of GFFE  tested on hDPCS were first examined by CCK-8 assay following 4-h culture . After iβ-Catenin, the transducer of Wnt signaling, has been reported to contribute to the maintenance of HF structures and is translocated to the nucleus which is important for the development and growth of hair follicles [a, which has been classified as a noncanonical Wnt family member, can also activate β-catenin signaling in the presence of the appropriate Frizzled receptor 4 [β-catenin signaling including DKK1, LEF1, Wnt5a, and β-catenin mRNA were investigated by means of RT-PCR.Wnt signals play a crucial role in the activation of bulge stem cells to progress toward hair formation and the induction of the anagen phase in hair growth which is required for the establishment of the hair follicle . The Wntollicles . It was ollicles . As a spollicles . DKK1 waollicles , 48. Wntceptor 4 . Genes rβ-catenin signaling, was reduced by 21%, 54%, and 67% in hDPCs treated with 0.008%, 0.016%, and 0.032% GFFE, respectively; the mRNA level of LEF1, a positive regulator of Wnt/β-catenin signaling, was increased by 66% (∗∗), 22% (∗), and 25% (∗) in hDPCs treated with 0.008%, 0.016%, and 0.032% GFFE, respectively; the mRNA level of wnt5a, a positive regulator of Wnt/β-catenin signaling, was increased by 162% (∗∗), 36% (∗∗), and 5%  in hDPCs treated with 0.008%, 0.016%, and 0.032% GFFE, respectively; the mRNA level of β-catenin, a positive regulator of Wnt/β-catenin signaling, was increased by 32% and 91% in hDPCs treated with 0.16% and 0.032% GFFE, respectively, but 0.008% GFFE decreased the expression of β-catenin (∗∗)  .β2 in DPCs, and these sequential events contributed to the shortening of the human hair cycle [β1 was regarded to regulate hair growth suppression by dose-dependently repressed hDPCs growth [β antagonists were effective in preventing catagen-like morphological changes and promoting elongation of hair follicles. In this study, GFFE decreased the expression of TGF-β1 in hDPCs. 0.008%, 0.016%, and 0.032% GFFE treatments showed 57%, 41%, and 64% decrease in TGF-β1 mRNA expression compared to NC, respectively (∗∗)  .VEGF mRNA is in the anagen phase and it is less expressed in the catagen and telogen phases in DPCs during the hair growth cycle [∗∗) and 19% (∗) greater VEGF mRNA expression compared to NC, respectively. However, 0.032% GFFE showed 6%  decreased VEGF mRNA expression compared to NC  to determine whether GFFE affects the hair-inducing ability of hDPCs. The above results suggested that GFFE could promote the hair-inducing ability of hDPCs. Next, we evaluated the effects of the GFFE on the protein expression and activity of Wnt5a protein level increased with all GFFE treatment groups. 0.008%, 0.016%, and 0.32% GFFE groups showed 66% (∗∗), 79% (∗∗), and 25% (∗) greater Wnt5a protein expression than NC, respectively , 67% (∗∗), and 81% (∗∗) attenuated TGF-β1 protein expression compared to NC, respectively (∗∗) increased VEGF protein level compared to NC, but VEGF protein level with 0.008% and 0.016% GFFE treatments showed 24% (∗∗) and 18% (∗) decreased level compared to NC, respectively  were shaved on the back when dorsal skin hair follicles are in telogen. GFFE or NC treatment was applied topically every day. In NC mice, only a few scattered pigmented spots were apparent at least until day 21. In contrast, both the GFEE  and 2% Minoxidil groups achieved hair regrowth and became remarkably grey after 7 days . FormatiThe morphology of the hair follicles was evaluated histologically to define the effect of GFFE on hair growth. The comparison and difference between NC, MXD, and GFFE groups in number, size, and depth of follicle were observed by histological photos , 3.68 mm (∗), 4.18 mm (∗∗), and 3.96 mm (∗), respectively. In day 21, the hair length of NC, MXD, 0.5% GFFE, 1% GFFE, and 2% GFFE was 4.93 mm, 7.86 mm (∗∗∗), 6.74 mm (∗∗), 7.91 mm (∗∗∗), and 7.60 mm (∗∗∗), respectively. In day 30, the hair length of NC, MXD, 0.5% GFFE, 1% GFFE, and 2% GFFE was 8.11 mm, 11.05 mm (∗∗), 10.21 mm (∗), 11.13 mm (∗∗), and 10.83 mm (∗), respectively (10 hair follicles were plucked from the mice's shaved area randomly from all groups, and the average length of each treatment was measured on day 14, day 21, and day 30. In day 14, the hair length of NC, MXD, 0.5% GFFE, 1% GFFE, and 2% GFFE was 2.43 mm, 4.12 mm (ectively .β-catenin signaling pathway activity while attenuating TGF-β1 synthesis activity in hDPCs and also found that GFFE up-regulated the protein expressions of VEGF and β-catenin and downregulated TGF-β. Moreover, GFFE promoted hair regeneration and repair in mouse models. Collectively, our results unveil a novel functional role of GFFE and strongly imply that GFFE can be developed as a herbal medicine for the treatment of hair loss diseases. In a future study, we will explore the specific constituents of antiremoval factors in GFFE as well as conduct GFFE in vivo and clinical trials to elucidate their application potential.In this study, GFFE showed strong hair growth-promoting activity. GFFE a was natural compound and has many pharmacological and physiological activities . The hai


Mechanistically, LINC01315 activated Wnt/β-catenin signaling. LINC01315 bound to the β-catenin promoter and activated its transcription. In rescue experiments, ectopic overexpression of β-catenin counteracted the inhibiting effector-triggered by LINC01315 deletion. In summary, this preliminary study brings new insights to the pathological significance of the LINC01315/Wnt/β-catenin signaling pathway in CRC.The pathological roles of long non-coding RNAs (lncRNAs) in colorectal carcinoma (CRC) have been corroborated. To date, the pathological contributions of LINC01315 in the epithelial-mesenchymal transition (EMT) property of CRC are still ambiguous. By silencing LINC01315, we disclosed that LINC01315 promoted the growth, metastatic characteristics, and the EMT of CRC cells   Colorectal carcinoma (CRC), one of the most frequent gastrointestinal malignancies, remains the primary trigger of malignancy-related mortality ,2. The mAnomalous activated Wnt-β-catenin classic signaling is essential in neoplastic cells metastasis and EMT program . Specifivia the miR-497-5p-sponging role ,26P < 0.nge| > 1 ). The pange| > 1 ). We evange| > 1 ). Notablnge| > 1 ). Comparnge| > 1 ). Finall.crg.eu) ). CollecTo figure out the biological role of LINC01315 in CRC progression, shLINC01315 was applied to knock down the endogenous LINC01315 expression in HCT116 and SW480 cells ). CCK-8 Wnt/β-catenin signal is implicated in cancer metastasis and EMT . The WesLncRNAs have been reported to regulate genes expression at the transcriptional level, the impact of LINC01315 on β-catenin was further examined. As shown in To confirm LINC01315-induced augment of CRC cells invasion and EMT was dependent on β-catenin. LINC01315-silenced CRC cells were transfected with pcDNA3.1-β-catenin plasmid to restore the expression of β-catenin ). In FigEmerging studies have focused on the cancer-specific lncRNAs and illuminated their imperative roles in cancer progression ,15. In tvia sponging miR-211 and then delays oral squamous cell carcinoma (OSCC) progression [Ectopic lncRNAs expressions have been implicated in cancer metastasis through the downstream effector molecules deregulation . LINC013gression . Patientgression . As a cegression . The waygression ,35.Although early studies show LINC01315 participates in the regulation of CRC cells’ mobility and invasion, the potential involvement of LINC01315 in CRC EMT has never been proposed before . DeregulMechanistically, LINC01315 directly binds to the promoter site of β-catenin, as demonstrated by the luciferase reporter gene assay. Further experiment with truncated constructs of β-catenin promoter indicates that LINC01315 binding site exists the sequence between the nucleotides −1756 and −1473 in the β-catenin promoter. Notably, our results showed that LINC01315 knockdown repressed CRC EMT and invasion, while β-catenin overexpression partially reversed the inhibition effects caused by LINC01315 knockdown.in vitro experiments. For the most part, in vitro experiments will still need to be followed up with in vivo experiments. In the follow-up work, we will proceed with a xenograft tumor model to explore the anti-metastasis of LINC01315 in vivo. In fact, the degree of β-catenin phosphorylation impacts the stability of β-catenin and directly alters the functions of the Wnt/β-catenin signal. Moreover, an increased Glycogen Synthase Kinase-3β (GSK-3β) stability diminishes the Wnt/β-catenin pathway via phosphorylating β-catenin [There are some deficiencies in the presented study. The results obtained are solely based on -catenin . Furtherin vitro. Further analysis indicated that LINC01315 exerts pro-oncogenic functions by promoting the transcriptional activation of β-catenin. This study yields new insights into the role of LINC01315/Wnt/β-catenin in CRC EMT.Collectively, we delineated that LINC01315 was exceptionally high in CRC and increased expression of LINC01315 predicted a poor prognosis in human CRC. Loss-of-function experiments demonstrated that LINC01315 fortified CRC cells migration, invasion, and EMT


Rusty pipe syndrome is a benign physiological condition that causes bilateral bloody discharge in lactating mothers. There is a bloody discharge from the nipples during lactation. It may lead to creating stress or anxiety in mothers, relatives as well as doctors. This is basically a harmless and self-limiting condition. There are various conditions for bloody discharge from nipples, like cracked nipples, mastitis, trauma, and ductal papilloma. This condition occurs during the first week of the lactation period. In this condition the color of breast milk changes to orange-brownish or rusty colored which seems similar to rusty brown water coming from a rusty iron pipe. Here we present the clinical image of rusty pipe syndrome, the rusty brown color which came from the small amount of blood from capillaries, mixes with colostrum or first breast milk. This condition is arising due to the high rate of vascularization of rapidly developing alveoli, which have delicate capillaries. Sometimes these capillaries get ruptured during the early lactation and ooze rusty breast milk. In this condition, feeding should be encouraged, and the mother should be counseled and aware. If the baby tolerates, feeding should continue. This condition is self-limiting and resolves within a week.


Glioblastoma is the most lethal form of brain tumor with a recurrence rate of almost 90% and a survival time of only 15 months post-diagnosis. It is a highly heterogeneous, aggressive, and extensively studied tumor. Multiple studies have proposed therapeutic approaches to mitigate or improve the survival for patients with glioblastoma. In this article, we review the loss of the 5′-methylthioadenosine phosphorylase (MTAP) gene as a potential therapeutic approach for treating glioblastoma. MTAP encodes a metabolic enzyme required for the metabolism of polyamines and purines leading to DNA synthesis. Multiple studies have explored the loss of this gene and have shown its relevance as a therapeutic approach to glioblastoma tumor mitigation; however, other studies show that the loss of MTAP does not have a major impact on the course of the disease. This article reviews the contrasting findings of MTAP loss with regard to mitigating the effects of glioblastoma, and also focuses on multiple aspects of MTAP loss in glioblastoma by providing insights into the known findings and some of the unexplored areas of this field where new approaches can be imagined for novel glioblastoma therapeutics. Glioblastoma multiforme (GBM) is the most common and lethal form of brain tumors with a median survival time of approximately 15 months after diagnosis . Previoude novo purine synthesis and provides an opportunity to specifically target cancer cells. Secondly, MTAP loss results in the accumulation of MTA and this inhibits the activity of several enzymes, including protein arginine methyltransferase 5 (PRMT5). PRMT5 is a regulatory protein critical for multiple processes such as genome organization, cell cycle regulation, and stem cell differentiation . SA. SA46]. IDE397 is another MAT2A inhibitor that is currently being evaluated in a clinical trial (NCT04794699). IDE397 is a differentiated small molecule inhibitor with great potential for MTAP-deleted cancer patients. IDE397 has demonstrated single-agent anti-tumor activity across various solid tumors including non-small cell lung cancer, gastric and bladder cancer among others.Besides AG-270 and IDE397 which inhibit MAT2A, in the clinical trial NCT03666988, Fedoriw et al. have shown GSK3368715 as a potential type I PRMT inhibitor . The defFalchook et al. have used PRT811, another PRMT5 inhibitor for treating advanced gliomas. PRT811 is a molecule which worked as a potent, selective brain penetrant PRMT5 inhibitor in animal models of brain tumors . In the AMG 193 is another PRMT5 inhibitor which preferentially binds with MTA-bound state of PRMT5 which is abundant in MTAP-deficient tumors. Clinical trial NCT05094336 uses AMG 193 to check efficacy in advanced MTAP-null solid tumors .TNG908 is also a PRMT5 inhibitor and is being orally administered in the clinical trial NCT05275478 to treat MTAP-deleted advanced solid tumors. TNG908 can bind with PRMT5-MTA complex leading selective inhibition of PRMT5 in MTAP-deleted tumors [MRTX1719 is another inhibitor which targets PRMT5-MTA complex and inhibits PRMT5 activity in animal model of MTAP-deleted tumors . It is bMTA together with 6-thiogunaine (6-TG) provides selective treatment for cancers with MTAP loss . TherapeClinical trials having NCT identifiers NCT00062283 and NCT00075894 use anitibiotic l-alanosine as an inhibitor for MTAP-deficient tumor cells. L-alanosine inhibits purine synthesis by blocking de novo purine synthesis pathway . These tMTAP loss has shown potential to create novel GBM therapeutics according to a number of studies; however, Menezes et al.  suggest


ACOX1 depletion promotes CRC cell proliferation in vitro and colorectal tumorigenesis in mouse models, whereas ACOX1 overexpression inhibits patient-derived xenograft growth. Mechanistically, DUSP14 dephosphorylates ACOX1 at serine 26, promoting its polyubiquitination and proteasomal degradation, thereby leading to an increase of the ACOX1 substrate PA. Accumulated PA promotes β-catenin cysteine 466 palmitoylation, which inhibits CK1- and GSK3-directed phosphorylation of β-catenin and subsequent β-Trcp-mediated proteasomal degradation. In return, stabilized β-catenin directly represses ACOX1 transcription and indirectly activates DUSP14 transcription by upregulating c-Myc, a typical target of β-catenin. Finally, we confirmed that the DUSP14-ACOX1-PA-β-catenin axis is dysregulated in clinical CRC samples. Together, these results identify ACOX1 as a tumor suppressor, the downregulation of which increases PA-mediated β-catenin palmitoylation and stabilization and hyperactivates β-catenin signaling thus promoting CRC progression. Particularly, targeting β-catenin palmitoylation by 2-bromopalmitate (2-BP) can efficiently inhibit β-catenin-dependent tumor growth in vivo, and pharmacological inhibition of DUSP14-ACOX1-β-catenin axis by Nu-7441 reduced the viability of CRC cells. Our results reveal an unexpected role of PA reprogramming induced by dephosphorylation of ACOX1 in activating β-catenin signaling and promoting cancer progression, and propose the inhibition of the dephosphorylation of ACOX1 by DUSP14 or β-catenin palmitoylation as a viable option for CRC treatment.Metabolic reprogramming is a hallmark of cancer. However, it is not well known how metabolism affects cancer progression. We identified that metabolic enzyme acyl-CoA oxidase 1 (ACOX1) suppresses colorectal cancer (CRC) progression by regulating palmitic acid (PA) reprogramming. ACOX1 is highly downregulated in CRC, which predicts poor clinical outcome in CRC patients. Functionally,  Alterations of intracellular and extracellular metabolites caused by metabolic reprogramming have profound effects on gene expression, protein modification, cellular differentiation, and the tumor microenvironment5. Metabolic enzyme acyl-CoA oxidase 1 (ACOX1), a rate-limiting enzyme in peroxisomal fatty acid β-oxidation, catalyzes acyl-CoA conversion to enoyl-CoA6. ACOX1 preferentially oxidizes long or very long straight-chain fatty acids9, while the related enzymes ACOX2 and ACOX3 catabolize branched-chain fatty acids and intermediates involved in bile acid synthesis10. Knockout of ACOX1 promotes hepatocellular carcinoma in mice12, and overexpression of ACOX1 inhibits oral cancer progression13. In addition, ACOX1 acts as a target gene of mir-15B-5p to inhibit tumor cell metastasis14. These studies indicate the inhibitory role of ACOX1 in cancer15. However, the role of metabolic reprogramming caused by dysregulation of the metabolic enzyme ACOX1’s post-translational modification in colorectal cancer (CRC) remains elusive.Metabolic reprogramming is critical for malignant transformation and tumor initiation and progression7 and a dominant fatty acid in a high-fat diet16, has been shown to produce energy and regulate intracellular signaling molecules involved in the development of cancer17. Previous studies have identified that PA promotes metastasis in melanoma, breast cancer, and gastric cancer in a CD36-dependent manner19, and also promotes the growth of prostate cancer by activating STAT3 signaling20. Recent research has revealed that dietary metabolite PA alters transcriptional and chromatin programs by modulating H3K4me3 in oral carcinomas and melanoma21. Furthermore, PA can modify cysteine residues in a process termed palmitoylation24. Increasing evidence suggests that palmitoylation of proteins  affects protein functions and tumor progression27. Therefore, whether ACOX1-mediated PA reprogramming affects tumor progression by regulating protein palmitoylation remains unknown.Palmitic acid (PA), an ACOX1 substrate29. Studies have shown that β-catenin signaling is abnormally activated in more than 90% of patients with CRC30. Post-translational modifications  of β-catenin have been demonstrated to regulate β-catenin signaling34. In addition, emerging evidence indicates that PA complements the β-catenin signaling activity19. However, whether β-catenin could be palmitoylated by PA remains unclear.β-catenin signaling is essential for maintaining cell homeostasis and embryonic development and is related to tumor cell proliferation, apoptosis, invasion, stemness, and chemotherapy resistanceHere, we demonstrate that ACOX1 is significantly underexpressed in CRC through a systematic bioinformatics screen and propose that reprogramming of PA induced by dysregulation of ACOX1 post-translational modification promotes CRC progression by activating β-catenin signaling via PA-mediated β-catenin palmitoylation and stabilization.35 were analyzed in at least 1000 CRCs from various datasets, including The Cancer Genome Atlas (TCGA) CRC RNA-SeqV2, TCGA CRC RNA-Seq, and Gene Expression Omnibus (GEO)  was notably downregulated in CRCs  39 and AOM40-induced mouse CRC models  Fig. . Importaels Fig. . Given t41 datasets  containing 192 CRC tissues by IHC  in CRC cell lines , respectively  with each expressing a tagged version of a protein were lysed and immunoprecipitated, followed by MS to identify their biophysically interacting proteins)tor Fig. , and thetor Fig. . Subsequtor Fig. . A time-lls Fig. .Fig. 3DU44. Therefore, to explore whether DUSP14 promotes ACOX1 degradation via the ubiquitin-proteasome system, we transfected HEK293T cells with Myc-Ub, ACOX1, and DUSP14 wild type (WT) or DUSP14 mutant (DUSP14 Dead) with mutation of cysteine 111 to serine45, which damaged the phosphatase activity. Ubiquitination assays showed that DUSP14 WT (not DUSP14 Dead) overexpression markedly increased the polyubiquitination of ACOX1  mRNA levels but increased β-catenin abundance in HCT15 and RKO cells  was performed in the TCGA database. GSEA revealed that ACOX1 negatively correlated with Wnt signaling, but not other cancer-related signaling pathways    in the kb) Fig.  and the kb) Fig. . EctopicOX1 Fig.  and c-MyP14 Fig. . Chromatlls Fig. . Luciferlls Fig. .ACOX1 mRNA and protein levels were significantly decreased, whereas the mRNA and protein levels of DUSP14 were highly increased in intestinal adenomas of APCMin/+ mice . In addition, TCF/β-catenin not only stimulates gene transcription but can also repress it68. This is possibly mediated by directly recruiting repressive factors, such as Reptin or Fhit that associate with the TCF/β-catenin complex and thus repress β-catenin-mediated transcription70, which may explain the mechanism behind the repressive effect of the TCF/β-catenin complex on ACOX1 expression in this study. Furthermore, the E3 ubiquitin ligase and protein kinase of ACOX1, as well as the palmitoyl transferase and de-palmitoyl transferase of β-catenin have not yet been discovered. Another limitation of this study is that specific antibody recognizing phosphorylated S26 of ACOX1 has not yet been developed. We will conduct follow-up research to address the above concerns in the future. In addition, given that fatty acid β-oxidation in peroxisome and mitochondria share some common substrates (mainly some long-chain fatty acids)71, we speculate that mitochondria-mediated PA β-oxidation may also affect CRC progression.Although popular models suggest that β-catenin phosphorylation/ubiquitination should be inhibited by The results of our study have revealed that ACOX1 is a tumor suppressor and critical for the supervision of β-catenin signaling by regulating PA-mediated β-catenin palmitoylation and stabilization. We have also proposed that inhibition of the dephosphorylation of ACOX1 by DUSP14 or β-catenin palmitoylation may be a viable option for CRC treatment.2 incubator. For transfection, after growing to 70% confluence, cells were transfected using Lipofectamine 3000  or HighGene , according to the manufacturer’s instructions.Human HCT15, RKO, HCT8, SW620, HCT116, HIEC-6, and HEK293T cells were obtained from the American Type Culture Collection (ATCC). Cells were cultured in DMEM medium  supplemented with 10% fetal bovine serum  and 1% penicillin-streptomycin  at 37 °C in a 5% CON-Ethylmaleimide  and BMCC-biotin ((1-Biotinamido)-4-[4′-cyclohexanecarboxamido]hexane, C100222-0050) were purchased from Sangon Biotech. Human palmitic acid ELISA kits (MM-51627H2) were purchased from MeiMian . Anti-Flag agarose beads (23101) and Nu-7441 (503468-95-9) were purchased from Selleck . RNase A (CW2105) was purchased from CWBIO. All antibodies used in this study are indicated in Supplementary Table DUSP14, ACOX1, CTNNB1, and c-Myc coding sequences were amplified from HEK293T cDNA and cloned into pCMV-HA and pHAGE-CMV-MCS-PGK vectors. The human GSK3β and CK1 coding sequences were amplified from HEK293T cDNA and cloned into the pHAGE-CMV-MCS-PGK vector. The human ACOX1-TBE and DUSP14-RE were amplified from HCT15 gDNA and cloned into the pGL3-basic luciferase vector. The mouse ACOX1 coding sequence was amplified from mouse colon cDNA and cloned into pCDH-CMV-MCS-EF1-GFP+Puro vector. Mutations in the DUSP14, ACOX1, β-catenin, and Ubiquitin cDNAs were generated by overlap extension PCR. Deletion mutants from DUSP14 and ACOX1 were cloned into the pHAGE-CMV-MCS-PGK vector. Human DUSP14, CTNNB1, ACOX1, and mouse Acox1 shRNAs were designed and synthesized by RuiBiotech , subsequently annealed, and inserted into the pLKO.1-puro vector. All primers for construction are presented in Supplementary Table Proteasome inhibitor MG132 (HY-13259), Nutlin-3 (HY-50696), Cytarabine (HY-13605), BMS-536924 (HY-10262), AZ628 (HY-11004), and palmitic acid (HY-N0830) were purchased from MedChemExpress. Cycloheximide (R750107), Hydroxylamine , 2-BP (238422), and DAPI (D9542) were purchased from Sigma-Aldrich. iCRT14 (sc-362746) was purchased from Santa Cruz. A dual-luciferase reporter assay kit (DL-101-01) was purchased from Vazyme. All animal studies were approved by the Animal Care Committee of Sun Yat-sen University. All mice were maintained in micro isolator cages in the Experimental Animal Center of Sun Yat-sen University.38. Briefly, eight-week-old C57BL/6 mice were injected intraperitoneally with 10 mg/kg AOM (Sigma-Aldrich). After 7 days, the mice were given drinking water containing 2.5% DSS  for a week, followed by regular drinking water for 2 weeks. Then, the mice were fed with 2.5% DSS water for two rounds for 1 week and sacrificed on the 120th day.AOM/DSS-induced mouse CRC model was performed following previously described methodsAPCMin/+/DSS-induced mouse CRC model was performed following the previously described methods39. Briefly, the eight-week-old APCMin/+ mice were fed with 1.5% DSS water for 1 week and sacrificed on the 75th day.72. Briefly, lentivirus was produced using polyethyleneimine-mediated transfection of a second-generation packaging system in HEK293T cells. Supernatant containing lentivirus was harvested at 72 h after transfection and filtered using a 0.45-μm filter. The lentivirus containing supernatant was then mixed with concentrate solution (5% PEG8000 and 0.5 M NaCl) overnight and concentrated by centrifugation at 4 °C. Virus titers were determined by ELISA kit.Lentivirus production was performed following previously described methodsAPCMin/+/DSS-induced or AOM/DSS-induced CRC mice, the eight-week-old mice were randomly assigned to indicated groups. The control group of mice was treated with lentivirus-expressing Ctrl or shCtrl and the indicated group(s) was/were treated with lentivirus-expressing indicated protein or shRNA. Concentrated lentivirus was delivered intraperitoneally to indicated mice twice per week for 3 weeks55. On day 75 or 120, the indicated mice were sacrificed and their tumor burdens were evaluated. Tumors larger than 1 mm were counted and measured. Colon tissues were collected for RNA extraction, protein assays, and pathological studies.For administration of lentivirus to 3) = a × b2/2, where a is the length and b is the width.For PDX transplantation, patients-derived tumor tissues were subcutaneously transplanted into BALB/c nude mice. When the tumors reached a certain size, the subcutaneous tumor was dissected and decomposed into a tumor of ~1 mm, and then inoculated into the subcutaneous of BALB/c nude mice again. One week after transplantation, mice were randomly assigned to 2 groups and injected with lentivirus expressing Ctrl or Flag-ACOX1 every 2 days for 9 total times. At day 21, PDXs were collected for tumor volume measurement and IHC analysis. Tumor volumes were calculated by the equation V , and 5 × 106 HCT15 cells were suspended in 100 μL PBS and injected subcutaneously in the flanks of animals. Twelve days after transplantation, 2-BP (40 mg/kg) was delivered intraperitoneally to indicated nude mice once per day for 12 days. Tumor growth was monitored every two days for a total period of 24 days. Tumor volumes were calculated by the equation V (mm3) = a × b2/2, where a is the length and b is the width.For subcutaneous xenograft model, the experiment was performed following previously described methodsACOX1 transcript levels, and 24 pairs of these samples were used to analyze ACOX1, DUSP14, and β-catenin protein levels. Fifteen fresh CRC samples and matched normal adjacent tissues were used to analyze PA levels. 192 CRC samples were made into TMA to analyze indicated protein levels and overall survival. All samples were obtained from the Sixth Affiliated Hospital of Sun Yat-sen University. The diagnosis of CRCs was verified by histological review. Our study was approved by the Ethics Committee of the Sixth Affiliated Hospital of Sun Yat-sen University (2020ZSLYEC-232). All patients signed written informed consent forms before treatment.Forty-five early CRC samples and 51 normal adjacent tissues were used to analyze 43. Briefly, indicated lentiviral vectors were packaged in HEK293T cells. HCT15 or RKO cells were infected with lentiviruses in the presence of polybrene and were selected with 1 µg/mL puromycin for two weeks to obtain stable clones. The indicated protein expression in stable clones was validated by western blotting.Stable cell line construction was performed as described previously43. Briefly, total RNA was isolated from cells or tissues and subsequent reverse transcription was performed. qPCR was then performed with SYBR Green Supermix  using standard procedures. The β-catenin targets in this study were obtained from previous study73. All primer sequences used are listed in Supplementary Table GAPDH was used as an internal control.RT-qPCR assays were performed as described previously43. Briefly, cells transfected with the indicated plasmids were lysed in 1 mL Lysis buffer. For immunoprecipitation, the anti-Flag agarose beads were washed with 1 mL lysis buffer three times, and then 0.9 mL of cell lysate was added into the indicated group and incubated overnight at 4 °C. The next day, the agarose beads were centrifuged and the supernatant was discarded. Subsequently, the agarose beads were washed three times and mixed in a 2× SDS sample buffer. Lysate samples were boiled for 10 min and were analyzed by immunoblotting with the indicated antibodies.Co-IP and immunoblot analysis were performed as described previously75. Briefly, cells transfected with the indicated plasmids were lysed in 1 mL Lysis buffer containing 50 mM NEM, followed by centrifugation  and immuno-precipitation overnight with anti-Flag agarose beads. After washing three times, precipitates were divided evenly into two sections, with 1/2 used for the –HAM control, and the remaining 1/2 was used for the +HAM for 1 h at room temperature. The precipitates were gently washed once with Wash Buffer , and incubated with BMCC-biotin Buffer  for 1 h at 4 °C. Then the precipitates were gently washed two times again with Wash Buffer. After washing samples were analyzed by SDS-PAGE and blotting, palmitoylated β-catenin was detected with HRP-conjugated streptavidin .For detecting protein palmitoylation, the acyl-biotin exchange (ABE) method was used76. Briefly, cells were lysed in Lysis buffer, and 80 μL supernatant was saved as input. The remaining supernatant was used for ABE experiments. Then palmitoylated proteins were enriched using streptavidin agarose (Cytiva) with rotation overnight at 4 °C. Samples were centrifugated at 3000 rpm for 5 min. 80 μL supernatant was saved as output. Protein-bound streptavidin agaroses were washed three times with Wash Buffer and bound proteins were eluted with SDS loading buffer for 10 min at 95 °C. Samples were subjected to SDS-PAGE. The fraction of palmitoylated β-catenin was determined by western blotting and calculated based on the β-catenin protein level in input and output samples. β-actin was blotted as a loading control.Streptavidin pulldown-based quantification of palmitoylated protein was performed as previously describedCells were cross-linked in situ with 1% formaldehyde for 10 min, quenched with 0.125 M glycine for 5 min at room temperature, and lysed in SDS Lysis buffer. Total lysates were sonicated to smash chromatin DNA to a size range of 200–1000 bp. The supernatant was diluted 10 times in ChIP Dilution buffer and precleared with 50 μL agarose beads for 2 h at 4 °C. Then the supernatant was collected by centrifugation, and the indicated antibodies (2 μg) were added to the supernatant. Then, the mixture was rotated overnight at 4 °C. The next day, 50 μL agarose beads were added, and rotation was continued for 2 h at 4 °C. Subsequent de-crosslinked DNA was subjected to PCR analysis using specific primers listed in Supplementary Table 43. Briefly, HEK293T cells were transfected with the indicated plasmids and treated with 20 μM MG132 for 6 h before collection. The cells were then lysed in RIPA lysis buffer and denatured by heating at 95 °C for 5 min. Immunoprecipitation analysis was performed as described above. The samples were boiled for 10 min in SDS sample buffer and analyzed by immunoblotting with the indicated antibodies.Ubiquitination assays were performed as described previously43. Briefly, the cells were transfected with the indicated plasmids, and 36 h later, the cells were treated with cycloheximide  for the indicated time periods before collection. The cells were lysed and proteins were detected by immunoblotting with the indicated antibodies.Protein half-life assays were performed as described previouslyACOX1-TBE, DUSP14-RE, or indicated mutant and 50 ng Renilla luciferase reporter were transfected in triplicates into HCT15 or RKO cells. After 36 h, luciferase activities were determined by the Dual-Luciferase Reporter Assay System. The Renilla activity was used as an internal control.0.3 μg pGL3 vector expressing 77.For protein qualitative analysis, HEK293T cells were transfected with the Flag-ACOX1 plasmid, lysed in Lysis buffer, and immune-precipitated with anti-Flag agarose beads. After SDS-PAGE and Coomassie Blue staining of the Flag-ACOX1-associated complexes, the bands were cut, subjected to in-gel trypsin digestion, and dried. The protein composition and protein site modification were analyzed by MS according to the protocols described previously78. Proteins were identified by Firmiana, a one-stop proteomic data processing platform79. Briefly, Mascot  and Nation Center for Biotechnology Information (NCBI) Ref-Seq human proteome database (updated on 04-07-2013) were used in the identification and quantification processes. Peptides (FDR < 0.01) were selected, and the proteins that contain high-quality and unique peptides were considered qualified. Length of minimal peptide was seven amino acids. Label-free intensity-based absolute quantification (iBAQ) was used to quantify proteins80. Fraction of total (FOT) was defined as the iBAQ value per protein divided by the sum of all protein iBAQ values. The FOT value was multiplied by 105 for easy representation.For protein quantitative analysis of CRC samples, five fresh CRC samples and matched normal adjacent tissues were collected from the Sixth Affiliated Hospital of Sun Yat-sen University, and lysed in Urea Lysis buffer. Extracted proteins were subjected to LC-MS/MS  analysis according to the standard protocols80.For cell proteome analysis, HCT15 cells were treated with PA (100 μM) or DMSO for 72 h, and lysed in Urea Lysis buffer. Extracted proteins were further digested, purified and measured. One microgram of protein per sample was subjected to LC-MS/MS analysis according to the standard protocols81. Briefly, human intestinal normal epithelial cells were maintained with organoid culture advanced DMEM/F12 containing growth factors (100 ng/mL Noggin (Peprotech), 500 ng/mL R-spondin (Peprotech), 50 ng/mL epidermal growth factor (Peprotech) and 10 μM Y-27632 (Abmole)) and treated with PA (300 μM). After spheroid organoid formation for 5 days, organoids were photographed and measured in diameter.Organoid assays were performed as described previouslyColon samples were fixed and embedded in paraffin according to standard protocols. H&E staining was performed in paraffin-embedded sections using hematoxylin and eosin (Servicebio). The analysis of IHC was performed using indicated antibodies against ACOX1 (Abcam) and Ki67 (Servicebio). The IHC staining results were scored considering both the intensity of staining and the proportion of tumor cells with positive reaction. The intensity of staining was scored as follows: 0, negative; 1, weak; 2, medium; and 3, strong. The frequency of positive cells was scored as follows: 0, < 5%; 1, 1%–25%; 2, 25%–50%; 3, 50%–75%; 4, > 75%. Total score ranging from 0 to 12 was determined by multiplying the score of staining intensity and the score of positive area.P < 0.05) was performed using the unpaired or paired Student’s t-test or χ2 test. Data are presented as the means ± SD.For data analysis, GraphPad Prism 8.3.0, Microsoft Excel, and IBM SPSS Statistics 26 were used. Statistical significance (Supplementary Information


To explore the regulatory effect of ubiquitin specific protease 25 (USP25) on glioma cell proliferation, migration, invasion, and its underlying mechanism. β-catenin pathway. The coimmunoprecipitation was used to check Thankyrase 1 (TNKS1) ubiquitination levels.  The USP25-overexpressed and USP25-knockdown glioma cells were established on U251 and U87 cells, respectively. Glioma cell proliferation ability was evaluated by CCK-8 assay. Cell apoptosis and cell cycle were determined utilizing flow cytometry. The Transwell assay measured cell invasion with wound healing used for cell migration detection. Western blotting established key protein expression levels in the Wnt/β-catenin pathway key protein downregulation and upregulation, respectively. Glioma cell invasion, migration, and proliferation activity were dramatically inhibited in USP25-knockdown glioma cells and promoted in USP25-overexpressed glioma cells. TNKS1 ubiquitination level was knowingly increased in USP25-knockdown glioma cells and reduced in USP25-overexpressed glioma cells, suggesting TNKS1 ubiquitination levels were negatively regulated by USP25.  TNKS1 expression levels were found to be considerably repressed in USP25-knockdown glioma cells and elevated in USP25-overexpressed glioma cells, accompanied by Wnt/β-catenin through the deubiquitination on TNKS1. USP25 facilitated glioma cell invasion, migration, and proliferation by regulating Wnt/ Glioma, accounting for around 80% of central nervous system (CNS) primary malignant tumors, is the most widespread primary malignant intracranial brain tumor. Due to the high recurrence rate, drug resistance, and high mortality, rare progression has been achieved by the current treatments of radiotherapy and chemotherapy. In recent years, novel strategies have been proposed based on targeted molecular therapies. It is critical to conduct in-depth research on the pathological molecular mechanism of glioma and offer a theoretical basis for promising therapeutic targets.β-catenin signaling pathway is a vital part of adult development and homeostasis, and its abnormal regulation is informed to facilitate malignant tumor development and progression [β-catenin/Wnt or truncation mutation on adenomatous polyposis coli protein (APC) can induce the over activation of the Wnt/β-catenin signaling pathway, which further promotes the development of colorectal carcinoma, gastric carcinoma, and hepatoma [β-catenin pathway inhibitor development as a new anticancer therapy will be a major breakthrough in the field of malignant tumors [β-catenin signaling pathway, Axin is glycosylated by TNKS, and the glycosylated Axin induces the ubiquitination of TNKS and the degradation of the proteasome by acting on the ubiquitin E3 ligase RNF146 [β-Catenin will be downregulated by the stably expressed Axin, which results in the declined transcriptional activity of β-catenin. Mariotti et al. [β-catenin could be induced by the inhibition on TNKS (TNKS1 and TNKS2), which further reduced colorectal cancer cell Wnt signaling activity. Therefore, inhibition of TNKS is considered as a novel method for malignant tumor treatment by blocking the Wnt signaling pathway [The Wnt/gression . The muthepatoma , 3. Thert tumors , 5. Thane RNF146 . β-Cateni et al.  reported pathway , 11.β-catenin signaling pathway. USP25 exerts a vital part in human malignant tumor cell proliferation and migration [β-catenin signaling pathway regulation via mediating TNKS1 ubiquitination.Deubiquitination is a vital protein stability regulating cellular mechanism . A deubiigration , 15. TheTherefore, in the present study, we investigated the effect of USP25 on the level of ubiquitination of TNKS1 and on the function and mechanism of glioma cell proliferation, invasion, and migration by interfering with the expression of USP25 in human glioma cells U87 and U251.2 and 37°C.U251 (Art. No. CL-0237) and U87 cells (Art. No. CL-0238) were bought from Procell . The cells were cultured in DMEM completed medium  supplemented with 10% fetal bovine serum  under the condition of 5% COTo knockdown or overexpress USP25 in glioma cells, USP25-siRNAs, and its control (siRNA NC), USP25 overexpression vector pcDNA3.1-USP25 (OE) and control pcDNA3.1-NC (OE NC) were designed and synthesized by General Biol. Inc. . The above interfering fragments or vectors were transfected into U87 and U251 cells, respectively, using Lipofectamine 3000 . After 48 h of transfection, the cells were collected for subsequent assays. The sequences for siRNAs were illustrated in β-catenin , phosphorylation-β-catenin , and GAPDH , respectively. After incubation with the secondary antibody  for 1.5 h, the membrane was hatched with the enhanced chemiluminescence (ECL) solution , followed by the ImageJ software quantification.Following extracting proteins from tissues and cells, quantification was performed on proteins, which were loaded onto 12% SDS-PAGE. Following separating for 1.5 h, proteins were transferred onto the PVDF membrane  and using 5% skim milk for blocking, followed by adding the primary antibody against TNKS1 , Cyclin D1 , C-myc , ΔΔCt− method was utilized to determine the normalization of gene expression completed with β-actin. Cell total RNA isolation was conducted with the TRIzol© reagent , then transcribed into cDNAs utilizing the TaqMan miRNA reverse transcription kit . The ABI 7900 real-time PCR machine was applied to conduct the PCR reaction using the SYBR® Green Real-time PCR Master Mix . The 23/well) were planted on 96-well plates for 24-hour incubation. The culturing medium supplemented with 10% CCK-8 solution  was then replaced. Finally, after 2 h, 570 nm absorbance was quantified with the use of the microplate reader .In brief, after digesting cells using 0.25% pancreatin, cells .4/well) were planted on the insert upper chamber . Medium comprised of 1% FBS was included in the lower chamber before 37°C incubation for 24 h. Then, the membrane upper surface was eliminated, and 0.1% crystal violet was utilized to stain lower chamber cells. Lastly, images were taken using the inverted microscope .Cells  and antibodies or control lgG were utilized to perform immunoprecipitation at 4°C for 4 h. After washing the beads three times with 1 ml of lysis buffer, the immuno-complexes underwent Western blot assay.Prechilled phosphate-buffered saline (PBS) was used to wash the transfected cells and lysed. After centrifugation, 50 t-test. Disparities between groups were evaluated with the one-way ANOVA method. P < 0.05 was deemed a considerable difference in this study.Data gained were presented as mean ± standard deviation (SD) with the GraphPad software used for analyzing. Disparities between the two groups were analyzed with the application of the Student's P < 0.05). Compared to the USP25 OE NC group (pcDNA3.1-NC transfected cells), USP25 was dramatically upregulated in the USP25 OE (pcDNA3.1-USP25 transfected cells) group (P < 0.05). Among the three siRNAs, USP25 expression in glioma cells transfected with siRNA-2 was the lowest. Therefore, siRNA-2 was chosen to knockdown USP25 in glioma cells in the subsequent experiments.As shown in Figures P < 0.05). On the contrary, compared to the OE NC group, a significantly higher expression of TNKS1 was detected in pcDNA3.1-USP25 transfected glioma cells (P < 0.05). These results revealed that TNKS1 expression was positively regulated by USP25 in the glioma cells.In P < 0.05). On the contrary, compared to the OE NC group, cell viability was dramatically elevated in glioma cells transfected with pcDNA3.1-USP25 (P < 0.05). Findings gained collectively proposed that proliferation of glioma cells was significantly facilitated by USP25.CCK-8 assay was employed for proliferation ability determination of U251 and U87 cells following different treatments. As indicated in P < 0.05). On the contrary, compared with OE NC, the migration ability was significantly increased after USP25 overexpression (P < 0.05), indicating that glioma cell migration ability was greatly facilitated by USP25.The wound healing assay was employed to assess glioma cell migration ability. P < 0.05). Compared to the OE NC group, invaded cell numbers dramatically declined in pcDNA3.1-USP25 transfected glioma cells (P < 0.05), revealing that glioma cell invasion ability was greatly facilitated by USP25.The invasion ability of glioma cells was determined by the Transwell assay. β-catenin, β-catenin, and c-myc levels expressed were greatly repressed in siRNA-USP25 transfected U251 cells or U87 cells (P < 0.05). However, after overexpression of USP25, all the above proteins were significantly upregulated (P < 0.05). This outcome indicates that USP25 activates the Wnt/β-catenin pathway in glioma cells.Figures Ubiquitination levels in U251 cells were evaluated by the Co-IP assay. Although novel techniques have been applied in the chemotherapy, radiotherapy, and surgery for glioma treatment in recent years, patient's survival continues to be poor due to the high degree of malignancy in glioma and the blurred interface between the lesion site and surrounding normal tissues.β-catenin were dramatically upregulated in glioma tissues when matched against normal brain tissue, implying that Wnt/β-catenin pathway may be a vital part of glioma as a classical signaling pathway positive regulator [β-catenin degradation and Wnt signaling pathway inhibition [β-catenin signaling pathway positively during mouse embryonic development progression, indicating that downregulation of TNKS1 might suppress the Wnt/β-catenin signaling pathway in glioma cells. Our prior data discovered that TNKS1 could facilitate the β-catenin nuclear transport, which further activates downstream target gene transcription expression, including Cyclin D1 and c-myc, to stimulate glioma cell proliferation and invasion [High expression of TNKS1 in multiple types of malignant tumors has been reported, involving the digestive tract, bladder, breast, and lung cancers –21. Our egulator . Lately,hibition . Feng ethibition  claimed invasion .USP25 is closely linked with malignant tumors, immune responses, and inflammation. For instance, USP25 is related to the endoplasmic reticulum-associated degradation, and the processing of amyloid precursor protein could be regulated by the acute ER stress-mediated ubiquitin dependent degradation , 28. USPβ-catenin signaling. In addition, the C-terminus of USP25 is found to interact with anchor protein repeats of TNKS1 directly, and X-ray crystal structure determination further characterizes the TNKS1 and USP25 interaction [β-catenin, Cyclin D1, β-catenin, and C-myc levels expressed in the Wnt pathway were positively regulated by USP25 in U251 and U87 glioma cells. Additionally, in U251 cells, TNKS1 ubiquitin level in the USP25-knockdown group was amplified, while TNKS ubiquitin levels in the USP25 overexpression group were considerably decreased, implying that TNKS1 ubiquitin levels in U251 cells were negatively regulated by USP25. The role of USP25-mediated ubiquitination of TNKS1 in regulating the Wnt pathway in glioma cells was further verified. To date, no therapeutic role has been found regarding USP25 in glioma. In contrast, this study provides a direct research basis for USP25 as a therapeutic target in glioma and its mechanism with TNKS1, and USP25 will become another significant discovery in glioma treatment.Recent studies have shown that Wnt signaling is positively regulated by USP25 mediating TNKS deubiquitination, while the degradation of TNKS1 could be facilitated by USP25, which contributes to Axin stabilization and the inactivation of Wnt/eraction . In the β-catenin regulation through the deubiquitination on TNKS1.All in all, the data gained revealed that USP25 facilitated glioma cell invasion, migration, and proliferation by Wnt/


The problem, however, is that it is difficult for consumers to calculate the CO2e value of food as it depends on several specifications such as the type of food, origin, etc. Could carbon labeling of food circumvent this problem and help consumers make more environmentally friendly choices?A conventionally grown kiwi from Spain or an organic pineapple from Ghana? Which is the more environmentally friendly option? Given that the production and distribution of food is responsible for about a quarter of our COIn an online experiment, 402 participants had to choose 20 food products from a fictitious online shop. The participants were randomly assigned to either one of three food labeling conditions  or the control condition.2e emissions, the purchase of more green food products and fewer red food products than in the control condition. The TLL outperformed the other two labels and was also the most accepted.The labeling conditions resulted in lower overall COThe carbon TLL is therefore a promising intervention to help consumers to not only choose more environmentally friendly foods, but also make a significant contribution to the fight against climate change. However, for its calculation, one needs several specifications for each product, such as how  and where  it was produced, how it was transported  and how it was preserved  to choose between different food options  or a color-coding system . One such ordinal rating label using a star rating system is the M-Check , but by its effective CO2e emissions in g . Hence, such labels can only be attached to certified products. One such certificate label is the abel see  which waabel see . The Cliheck see  which waheck see . Quantitsino see .2.2.grocery shopping. One of these is the study by ordinal plus quantitative rating label), a so-called Traffic Light Label (TLL), attached to 37 products with high turnover in a supermarket, influenced their purchase. The TLL color  of a product was determined by comparing the carbon footprint of that product  to the average carbon footprint of other products in the same category . For example, if the product’s carbon footprint was below this average, it received a green label. Whether this so-called relative color coding is the best method is unclear. Nevertheless, the authors found a substantial, but non-significant decrease of sales (from 32 to 26%) of high carbon footprint (black labeled) products and a non-significant increase of sales (from 53 to 57%) of green labeled products. However, when the labeled products were also the cheapest , a significant 20% increase in the purchase of green labeled products was observed. Although the effects in this study were small, the labels did appear to have an effect. Some more studies testing the impact of carbon labels on food choices are found in the restaurant area. Here too, carbon labels lead people to choose more environmentally friendly menus  performed better than the one without colors, which accordingly only showed the CO2e content in g . A similar result was observed in the study by The question arises why is the impact of carbon labeling rather small? If you have to choose between see also . AdditioIn summary, although research has shown that carbon labels can influence purchase decisions see also the reviews of carbon labels by 3.3.1.design elements be? Typically, consumers spend only a few seconds on food choices. For example, 5 s for a milk decision in a realistic scenario, a grocery store,  in a scene cluttered with a variable number of other distracting objects (other fruits). It has been shown in laboratory experiments that color is the object’s feature that can most efficiently guide attention to the target object , whereas green is associated with positive words  the healthiness of a food by weighing and adding up the different nutritional information such as fat, sugar, etc. Furthermore, it has been shown that, for example, the weighing itself can be slightly changed depending on the context  than on the back  and “in the name of sustainability” on a green background. Accordingly, we also used design elements from existing labels, such as the Climatop label. The second type of label is the TLL  or a control condition with no labeling. Regardless of the condition, participants were presented with 20 different food categories . Each category contained 6 products - depending on the condition with a specific type of label or not - from which participants had to choose 1 product by putting it in the shopping cart. Thereby we measured, the total carbon emissions of the shopping cart, the average rating of the products chosen in each food category and the number of red, orange and green products chosen. Furthermore, it was investigated which type of labeling is most accepted and which institution should initiate such a labeling regarding its trustworthiness.4.4.1.Mage = 26.05; ageSD = 6.45; 61.7% female) from ZHAW Zurich University of Applied Sciences and the greater area of Zurich took part in this computer-based online study. As an incentive, participants could enter a raffle for one of two iPads  or if a student of the ZHAW School of Applied Psychology receive course credit instead . All participants gave informed consent.Four hundred two participants aged 19 to 65 years old , only some products were supplemented with a label (see Green Foot condition), or all products were supplemented with a label (Star Rating and TLL conditions). Since the labels have already been described in detail in the introduction, there are just 2 additions. First, the total number of CO2e was only shown in the TLL condition, since neither “carbon reduced” labels  nor star rating labels  normally show these numbers. Second, the rating or color coding is relative, that is, per product category  products from 20 different categories: milk, cheese, citrus fruits, fish, coffee, pasta, rice, sausages, meat, etc. Foods were selected to cover the full range of everyday needs. The images (taken with permission from an online store of one of the largest Swiss retailers) were supplemented with the name, price, quantity as well as several COduct see . More pr4.3.At the beginning of the study participants had to imagine that they had to buy 20 food products for a housewarming party with friends from a new online shop . In doing so, they were reminded that as students, they have a limited budget. Note that nothing was said about sustainability, labels, etc. Participants were then guided through the shop or products by being shown 20 pages see , each coAfter the food shopping task, participants had to answer a series of questions. For example, in the label conditions, there were questions about the acceptance of the corresponding label such as “I consider the CO₂e label to be credible” . In addition, all participants had to answer the question as to how trustworthy2e (in g) emissions of the products purchased, that is, of those products that ended up in the shopping basket. The CO2e of a product was thereby calculated per 100 g. Second, we rated the products within each food category by the number of stars they would have received under the Star Rating label condition . This scale made it possible to assess if labels affect choices differently in each of the categories. This way of rating makes comparisons easier as the different categories have large differences in the mean and range of their carbon footprint . Third, we calculated the number of products purchased within the high (red), medium (orange), and low (green) footprint categories.Regarding the food shopping task, we first calculate the CO5.5.1.From the 449 participants that completed the study, 33 participants (7.3%) who needed less than 10 or more than 60 min to complete the study were excluded from the analysis. Furthermore, 14 participants (3.1%) who did not answer the control question correctly were excluded.5.2.5.2.1.planned contrast (under a one-way ANOVA) showed that the total g CO2e of the products bought in the no label (control) condition was significantly higher than in the label conditions , t(398) = 3.76, p < 0.001, d = 0.44. Tukey adjusted post-hoc tests showed a significant difference between the control condition and: the Star Rating label condition, t(398) = 2.73, p < 0.05, and the TLL condition, t(398) = 4.75, p < 0.001, but not the Green Foot label condition, t(398) = 1.81, p = 0.27. Within the label conditions, there was only a significant difference between the TLL condition and the Green Foot Label condition, t(398) = 3.01, p < 0.05  = 14.15, p < 0.001, 2η = 0.10, as well as a category, F = 41.51, p < 0.001, 2η = 0.09, but no significant Condition × Category interaction, F = 1.07, p = 0.33. Tukey adjusted post-hoc tests showed again a significant difference between the control condition and: the Star Rating label condition, t(398) = 3.86, p < 0.001, and the TLL condition, t(398) = 6.30, p < 0.001, but not the Green Foot label condition, t(398) = 2.23, p = 0.16. Within the label conditions, there was only a significant difference between the TLL condition and the Green Foot Label condition, t(398) = 4.16, p < 0.001 . These results are thereby in line with the results of overall CO2e emission (in g) calculations and show that labels have an effect on choice. Though the labels had no differential effect per category (no interaction), there are differences between the categories (see A repeated measure ANOVA showed a significant main effect for condition (labels and no label), ries see . However5.2.3.Do labels affect product purchases (compared to the control group) differently in the red (high), orange (medium), or green (low) footprint categories, and if so, how see also ? To inveχ2 (6) = 115.75, p < 0.001, R2 = 0.02, indicating a differential label effect regarding the purchase of green, orange, and red products. The subsequently calculated planned contrasts, for example, showed that all label conditions led participants to purchase more green products than in the control condition (Star Rating label condition: t(8) = 5.44, p < 0.01; TLL condition: t(8) = 8.91, p < 0.001; Green Foot label condition: t(8) = 4.58, p < 0.01). Within the label conditions regarding green choices, there was a significant difference between the TLL condition and Green Foot condition, t(8) = 4.54, p < 0.01, and the TLL condition and the Star Rating label condition, t(8) = 3.65, p < 0.05, but not between Star Rating condition and Green Foot label condition, t(8) = 0.84, p = 0.83. However, in the orange category, there were no differences in products purchased between the label conditions and the control condition, or between the label conditions themselves . Finally, the Star Rating label condition, t(8) = −3.75, p < 0.05, and the TLL condition, t(8) = −8.79, p < 0.001, but not the Green Foot label condition, t(8) = −2.20, p = 0.21, made participants buy less red products compared to the control condition. As regards the green products, differences between the labels were significant between the TLL condition and the Star Rating condition, t(8) = 5.26, p < 0.01, and the TLL condition and the Green Foot label condition, t(8) = 6.77, p < 0.001, but not between the Star Rating condition and the Green Foot label condition, t(8) = 1.57, p = 0.44. Accordingly, the labels, especially the TLL, had a positive effect in the green (more purchases) and in the red (less purchases) footprint category  = 5.06, p < 0.01, η2 = 0.032. Tukey adjusted post-hoc tests showed significant differences between the Star Rating label  and the TLL , t(304) = 2.83, p < 0.05, and the TLL and the Green Foot label , t(304) = 2.70, p < 0.05, but not between the Star Rating and the Green Foot label, t(304) = 0.11, p = 0.99. Overall, participants’ label acceptance was highest for the TLL, although the effect is rather small. The translated questions and means are shown in The overall consistency of the questions was good . A calculated one-way ANOVA showed a significant main effect for condition , 5.3.2.F = 127.61, p < 0.01, η2 = 0.29. Participants regarded a carbon label awarded by the State  and the WWF  as most trustworthy. Tukey-adjusted post-hoc tests found no difference between these two institutions, t(306) = 2.50, p = 0.13. Comparably , lower values of trustworthiness could be found for the federation of Swiss food industry , the EU , private companies  and international corporations . For a graphical representation see There was a significant effect regarding trustworthiness of the controlling institution, 6.carbon labels make consumers choose more environmentally friendly food products, especially under the TLL, an ordinal plus quantitative rating label. The TLL resulted in the largest reduction in 2e emissionsoverall CO (in g), shown by the purchase of more green  and less red  products. Thus, these results are consistent with those of existing research (see Green Foot (a certificate label) and also the Star Rating label  showed similar, albeit weaker, effects. Finally, TLL affected choice in all food categories and was also the most widely accepted by participants. Unlike other studies, in this study, not only was the effectiveness of multiple carbon labels (3 labels) tested simultaneously, but they were also tested among a broader range of products instead of just one, such as coffee or tomatoes  of a product. Alternatively, you could do this per serving size or package size. For example, 100 ml of wine, which is also considered a serving size in Switzerland, has about 360 g CO2e, whereas 100 ml of beer has only 115 g CO2e . This is because specifying by portion size has the disadvantage that non-standard portion sizes can easily be reduced by the retailer to make the values turn out better. This could also happen with carbon labels by offering, for example, smaller package sizes. People would then probably buy several small packages, which is no better than buying one large package. Out of curiosity, we ran our analysis again, taking package sizes into account. For example, if a participant chose the 150 g goat cheese, the value of 1,002 g CO2e instead of 668 g CO2e was newly included in the calculation. The label effects found were similar, indicating that the effect of carbon labels per 100 g is maintained even when differences in package size are considered  and the appropriateness of the sample . Another limitation could be that we did not record whether participants understood the meaning of the different terms. For example, Overall, this study adds to the existing carbon label literature by testing in a shopping task, three different label formats, whereby two have to our knowledge not been empirically tested so far (carbon certificate label and rating label without color). The study underpins the findings of other authors that carbon TLLs are more accepted and more effective than other labels tested so far. We also identified research gaps, such as a lack of research about the influence of the reference frame on TLL’s effectiveness, or a lack of research about negative carbon labels. Nevertheless, well designed carbon labels can influence decision making and lead consumers to carbon friendlier food choices.swen.kuehne@zhaw.ch.The datasets presented in this article are not readily available because only aggregated data are shared. Requests to access the datasets should be directed to Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. The patients/participants provided their written informed consent to participate in this study.SK and ER: made an equal substantial, direct, and intellectual contribution in all stages of the work. LLV and MB: were involved in the design and programming of the study. All authors contributed to the article and approved the submitted version.Open access funding provided by Zurich University of Applied Sciences (ZHAW).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.