Figure 7

AngII inhibited autophagy while PP2Cα knockdown activated it via ULK1. (A) Representative western blots showing effects of AngII and PP2Cα siRNA on markers of autophagy. (B) AngII increased p62 accumulation (indicative of an inhibition of autophagy) and knockdown of PP2Cα prevented p62 accumulation. Specificity of the rescue was verified via utilization of two PP2Cα siRNAs, as indicated. All other data were generated from experiments utilizing PP2Cα siRNA ‘A’. (C) LC3-II conversion was increased with AngII, but increased further by PP2Cα knockdown. (D) PP2Cα siRNA increased expression of the autophagy marker Beclin-1. (E) AngII reduced expression of the lysosome marker LAMP1, but this reduction was not restored by PP2Cα siRNA. (F) AngII decreased activating ULK1 phosphorylation, which was prevented by PP2Cα siRNA. (G) AngII increased inhibitory ULK1 phosphorylation, which was prevented by PP2Cα siRNA. (H, I) There was no transcriptional regulation of p62 (H), or LC3A (I) with either AngII or PP2Cα siRNA. (J) Representative immunohistochemical images showing the AngII-mediated increase in p62-positive punctae, and the prevention of that increase via knockdown of PP2Cα, showing inhibition of autophagy with AngII and restoration of autophagic flux with PP2Cα knockdown. (K) Representative immunohistochemical images showing PP2Cα siRNA increased autophagosome number as determined by LC3-positive punctae. n =4-20, Mean ± SEM, *P <0.05, **P <0.01, ****P <0.0001 (Saline scrambled vs. AngII scrambled), +P <0.05 (AngII scrambled vs. AngII PP2Cα siRNA).