Fig 2: Intervening autophagy flux in hNSCs in TBI brain extracts at different phases. Markers of autophagy flux and the WNT/CTNNB pathway were detected (A). Related expression of proteins and mRNAs revealed that rapamycin (50 nM) could activate autophagy flux in the acute phase and 3-MA (2 µM) could inhibit autophagy flux in the subacute phase (B,C). The activation of the WNT/CTNNB pathway showed a corresponding change in the autophagy flux (B,C). The black circle in B,C represented the individual value of each sample. *: p < 0.05; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001; ns: p = 0.05.

Fig 3: Usp26 deficiency impairs osteoblastic differentiation of MSCs by decreasing ß-catenin.A Silver stained SDS-PAGE image of USP26-binding proteins pulled down by USP26 antibody in the supernatant of MSCs lysates. B The accession number, theoretical molecular mass, and the number of non-redundant peptides of ß-catenin identified by liquid chromatography–tandem mass spectrometry. C Co-immunoprecipitation of USP26 with endogenous ß-catenin in mouse MSCs. D Representative of ß-catenin IHC staining in femurs of 5-month-old Usp26-/- mice and their WT littermates (n = 5). Red and black bars represent 25 and 100 µm, respectively. E Western blot analysis of ß-catenin in MSCs obtained from Usp26-/- and littermate controls with or without osteoblastic differentiation. F Western blot analysis of ß-catenin in Usp26-/- MSCs with or without Ctnnb1 overexpression. G, H Osteoblastic genes expression, ARS and ALP staining of Usp26-/- MSCs with or without Ctnnb1 overexpression. I The schematic graph reflects that decreased protein of ß-catenin was responsible for impaired osteoblastic differentiation of Usp26-/- MSCs. J, K Co-immunoprecipitation of USP26 with ectopically expressed Flag-tagged ß-catenin in 293 T cells. L Overexpression of Usp26 decreases the level of ubiquitinated ß-catenin. M Western blot analysis of the protein level of ß-catenin in 293 T cells with or without Usp26 overexpression and treated with cycloheximide (CHX) for indicated time intervals. N The schematic graph reflects the underlying mechanisms of USP26 in decreasing ß-catenin degradation by reducing the level of ubiquitinated ß-catenin in 293 T cells. *P < 0.05, **P < 0.01, ***P < 0.001. P values were analyzed by one-way ANOVA. All data are representative of two to three independent experiments.

Fig 4: Effects of HQT on differential protein expression in CAC mice. (A) Western blot analysis of WNT3A?Cdh1?Ctnnb1?GSK3ß?Ccnd1 and CDK4 in mice colon tissues. (B) Statistical analysis of WNT3A?Cdh1?Ctnnb1?GSK3ß?Ccnd1 and CDK4 gray values. Data are represented as the mean±SD (n = 3, ¯x±s). *p<0.05, **p<0.01 vs. normal control; #p<0.05, ##p<0.01 vs. model control. C: Normal control; M: Model control; H: HQT control.

Fig 5: GLS1 regulates CSC properties via ROS/Wnt/ß-catenin signaling. Determination of cellular ROS in HCCLM3 cells. Cells were cultured with glutamine deprivation or 10 µM 968 for 48 h, and ROS was detected using DCFH-DA by flow cytometry (a) or confocal (b). H2O2 was used as positive control. Scale bars: 100 µm. (c) Colony formation assay was performed on HCCLM3 cell line treated with indicated concentration of H2O2. (d) H2O2 treatment suppressed the expression of KLF4, c-MYC, and ß-catenin. (e) Simplified schematic of the glutamine metabolism pathways. (f) HCCLM3 cells were assayed for colony formation in complete DMEM medium or without glutamine or in the presence of 10 µM 968 alone or together with 2 mM DM-a-KG or 4 mM NAC. (g) HCCLM3 cells were treated with 5 µM 968 for two weeks, then metabolomic analysis was performed to screen for the differentially expressed metabolites. (h) Genes associated with GLS1 were analyzed by gene set enrichment analysis (GSEA) based on TCGA dataset. Wnt/ß-catenin pathway was enriched. (i) HCCLM3 was treated with the indicated concentration of SKL2001 for 24 h, then GLS1 mRNA expression was examined. (j) HCCLM3 cells were treated with the indicated concentration of SKL2001 for 48 h (upper panel) or treated with 10 µM SKL2001 for 12, 24, and 48 h (lower panel), then GLS1 protein expression was measured. (k) HCCLM3 cells were treated with different concentration of 968 for 24 h. The mRNA levels of ß-catenin (CTNNB1) and its target genes (CCND1, BIRC5, BCL2, and AXIN2) were determined by q-PCR. (l) HCCLM3 cells were pretreated with 10 or 20 µM 968 for 24 h, then treated with SKL2001 for another 24 h. Cells were harvested and fractionated into cytoplasmic and nuclear fractions, thereafter the expression of GLS1, ß-catenin protein levels were determined by Western blot. GAPDH and lamin B1 were detected as fractionation and loading controls. (m) SMMC-7721 cells with stable knockout of GLS1 by CRISPR/Cas9 was treated with SKL2001 for 24 h, then cytoplasmic and nuclear fractions were prepared and subjected to Western blot with anti-GLS1 and anti-ß-catenin antibodies. Data are presented as mean ± SD. ****, p < .0001.