Fig 2: A schematic diagram illustrating the role of APE1 in activating and stabilizing YAP1. Exposure of cells to acidic bile salts (ABS) induce APE1-dependent increase in YAP1, leading to its activation and translocation into the nucleus. Mechanistically, APE1 stabilizes YAP1 by binding to β-TrCP thereby disrupting YAP1-β-TrCP interaction and preventing YAP1 polyubiquitination and degradation

Fig 3: APE1 regulates YAP1 stability through β-TrCP ubiquitinase. A-B FLO-1 (A) and OE33 (B) cells with or without APE1 transient knockdown were treated with cycloheximide (CHX, 100 µg/mL) for the indicated timepoints. The levels of APE1 and YAP1 were determined using western blot. β-actin was used as a loading control. C FLO-1 and OE33 cells with or without APE1 transient knockdown were treated or not with MG132 (10 µM) for 16 h. The levels of APE1 and YAP1 were evaluated using western blot. β-actin was used as a loading control. D Western blot was used to analyze APE1 and β-TrCP expression following transient APE1 knockdown (si-APE1) in FLO-1 and OE33 cells. β-actin was used as a loading control. E FLO-1 and OE33 cells were transfected with wild type APE1 (flag APE1), immunoprecipitated with flag antibody and immunoblotted with β-TrCP and APE1 antibodies. F-G Representative images of proximity ligation assay for APE1 and β-TrCP in FLO-1 and OE33 cells (F) and YAP1 and β-TrCP in OE33 cells with and without APE1 knockdown (G). DAPI (blue) was used for nuclear staining. H In vitro ubiquitination assay in FLO-1 and OE33 cells transfected with HA-ubiquitin plasmid then treated or not with APE1 redox inhibitor E3330 (100 µM, overnight) followed by MG132 treatment (10 µM for 6 h). YAP1 antibody was used for immunoprecipitation and samples were then blotted with anti-ubiquitin and anti-YAP1

Fig 4: APE1 mediates ABS-induced YAP1 activation. A Representative immunofluorescence images of APE1 (red) and YAP1 (green) in FLO-1 cells exposed to ABS (200 µM, pH 4, 20 min) followed by 3 h recovery with or without APE1 knockdown. DAPI (blue) was used for nuclear staining. B Quantification of immunofluorescence stain intensity using ImageJ. C Cytosolic and nuclear fractions from FLO-1 (left) and OE33 (right) cells transfected with si-Ctrl or si-APE1 and treated with ABS (200 µM, pH 4, 20 min) followed by recovery in complete media for indicated timepoints were evaluated by western blot for the levels of APE1 and YAP1. β-tubulin and p84 were used as a loading control for cytosolic and nuclear fractions, respectively. D 8xCTIIC luciferase reporter assay was used to measure YAP1 transcriptional activity in FLO-1 and OE33 cells transfected with si-Ctrl or si-APE1 and exposed or not to ABS treatment (200 µM, pH 4, 20 min) followed by 3 h recovery. The luciferase reporter activity values were normalized to Renilla expression levels. Values are represented as mean ± SEM of three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001

Fig 5: ABS enhances sphere formation and induces YAP1 through APE1. A Representative bright field images of OE33 spheres derived from control cells (UT) or cells repeatedly exposed to ABS (200uM, pH5.5) for 20 min per day for 14 days (rABS). Quantification of the number and diameter of spheres is also presented. B Western blot analysis of APE1 and YAP1 in OE33-derived spheres with or without repeated ABS. C Representative bright field images of spheres derived from repeated-ABS-treated OE33 cells with or without E3330 (50 µM) treatment. Quantification of the number and diameter of spheres is also presented. D Western blot analysis of APE1 and YAP1 in spheres derived from repeated-ABS-treated OE33 cells with or without E3330 (50 µM) treatment. E Representative bright field images of spheres derived from repeated-ABS-treated OE33 cells with or without transient APE1 knockdown. Quantification of the number and diameter of spheres is also presented. F–H Representative immunofluorescence images of spheres derived from repeated-ABS-treated OE33 cells with or without E3330 (50 µM) treatment showing APE1 (red) and YAP1 (green) staining in (F), CTGF (green) in (G) and CD44 (green) in (H). DAPI (blue) was used for nuclear staining. Quantification of immunofluorescence intensity by ImageJ is also shown. Values are represented as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001