Fig. 6
NOX4 regulates PKM2 stability through PCAF-dependent acetylation and lysosomal mechanisms. a PKM2 was immunoprecipitated using PKM2 antibodies in shVec or shNOX4 786-O cells and acetylation assessed using acetylation antibodies (noted by the arrow). PKM2 pull down was validated (lower panel). b (Left panel) PKM2 expression in shVec and shNOX4 786-O cells treated (+) or not (−) with 5 mM 3-MA for 24 h. (Right panel) Quantitation of b where ±S.E.M. *p < 0.05 is compared to shVec without (−) 3-MA and # p < 0.05 compared to shNOX4 without (−) 3-MA. c Apoptosis was assessed in shNOX4 786-O cells pre-treated (+) or not (−) with 3-MA followed by exposure (12–14 h) with etoposide or buffer (−) where ±S.E.M. ***p < 0.001 is compared to non-treated shNOX4 (−) and ### p < 0.001 compared to shNOX4 etoposide. d shNOX4 786-O cells were transfected with scrambled or siRNA specific for PCAF or p300. After 24 h, the cells were treated (+) or not (−) with 20 µM etoposide (12–14 h). The results are expressed as the means where ±S.E.M. ***p < 0.001 is compared to shNOX4 (scr) control without Etop and ### p < 0.001 compared to shNOX4 (scr) + Etop. NS represents non-significance. e In parallel to d, PKM2 expression was assessed and knockdown of PCAF and p300 were validated. GAPDH was used as a loading control. f (Upper panel) shNOX4 786-O cells were transfected with PKM2 acetylated mutant (K305R), using lipofectamine, for 24 h and subsequently exposed to 20 µM etoposide. Apoptosis was evaluated. The results are expressed as the means where ±S.E.M. *p < 0.05 is compared to non-treated Vector (Vec) control and ## p < 0.01 compared to shNOX4 cells transfected with Vec control + Etoposide. (Lower panel) Overexpression of PKM2 (K305R)-Flag acetylation mutant was verified by immunoprecipitation of TCL using Flag (anti-mouse) and western blot analysis using Flag (anti-Rabbit) antibody. g PKM2 expression was assessed by western blot analysis in 786-O cells stably silenced by NOX4 or shVector control with transfection of the ATP mutant NOX4 (K540A). h PKM2 expression was examined in 786-O with or without VHL, transfected (+) or not (−) with ATP mutant NOX4 (K540A). Actin was used as loading control. Statistics are expressed as the means using one-way ANOVA with Tukey’s post hoc test
