Fig. 4

ABS-induced YAP1 activation depends on APE1 redox activity. A FLO-1 and OE33 cells were pre-treated or not with NAC (200 µM, 2 h) then exposed to ABS (200 µM, pH 4, 20 min) and allowed to recover for 3 h in complete media. Western blot analysis was used to evaluate APE1 and YAP1 protein levels. β-actin was used as a loading control. B FLO-1 and OE33 cells with transient knockdown of APE1 using si-APE1 were reconstituted with wild type APE1 (flag APE1) or APE1 redox mutant (C65A). Western blot was used to analyze the protein levels of APE1 and YAP1. β-actin was used as a loading control. C FLO-1 and OE33 cells were pre-treated or not with APE1 redox inhibitor E3330 (100 µM, overnight) then exposed to ABS (200 µM, pH 4, 20 min) and allowed to recover for 3 h in complete media with or without E3330. Western blot analysis was used to evaluate APE1 and YAP1 protein levels. β-actin was used as a loading control. D FLO-1 and OE33 cells were transfected with 8xCTIIC luciferase reporter and Renilla. Cells were then pre-treated or not with APE1 redox inhibitor E3330 (100 µM, overnight) then exposed to ABS (200 µM, pH 4, 20 min) and allowed to recover for 3 h in complete media with or without E3330. The relative 8xCTIIC luciferase reporter activity was measured and reported as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001. E–F 8xCTIIC luciferase reporter assay was used to measure YAP1 transcriptional activity in FLO-1 (E) and OE33 (F) cells transfected or not with wild type APE1 (flag APE1) or APE1 redox mutant (C65A) with or without exposure to ABS (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. * P < 0.05; ** P < 0.01; *** P < 0.001