BRG1-dependent Wnt/β-catenin pathway activation promotes tubular senescence through autophagic inhibition
(A,B) Western blot analysis demonstrated that BRG1-mediated autophagy inhibition was reversed by pharmacologic inhibition of Wnt/β-catenin signalling. mTECs were transfected with BRG1 expression vector (pReceiver-M14-BRG1), followed by stimulation with ICG-001 (5 µM) for 48 h. Representative Western blot (A) and quantitative data on the relative abundance of LC3A/B-II, SQSTM1, and Beclin-1 proteins in different groups (B) are presented. *P<0.05, **P<0.01, ***P<0.001 versus control group; #P<0.05, ###P<0.001 versus pReceiver-M14-BRG1 transfection alone (n=3). (C–G) Western blots analysis showed that the activation of autophagy by RAPA blocked BRG1-induced p16INK4a and p21 proteins expression. mTECs were transfected with pReceiver-M14-BRG1, followed by treatment with or without ICG-001 (5 µM, 48 h) and RAPA (200 nM, last 2 h) as indicated. Representative Western blot (C) and quantitative data on LC3A/B-II (D), SQSTM1 (E), p16INK4a (F), and p21 (G) are presented. *P<0.05,**P<0.01, ***P<0.001 versus control group; #P<0.05, ##P<0.01, ###P<0.001 versus pReceiver-M14-BRG1 transfection alone (n=3). (H–L) Western blot analysis showed that autophagy inhibition by Baf A1 eliminated the inhibition of BRG1-induced p16INK4a and p21 protein expression by ICG-001. mTECs were transfected with pReceiver-M14-BRG1, followed by treatment with or without ICG-001 (5 µM, 48 h) and Baf A1 (5 nM, last 2 h) as indicated. Representative Western blot (H) and quantitative data on LC3A/B-II (I), SQSTM1 (J) p16INK4a (K), and p21 (L) are presented. *P<0.05,**P<0.01, ***P<0.001 versus control group; #P<0.05, ##P<0.01, ###P<0.001 versus pReceiver-M14-BRG1 transfection alone. &P<0.05, &&P<0.01, &&&P<0.001 versus pReceiver-M14-BRG1 in the presence of ICG-001 (n=3). (M,N) Western blot analysis demonstrated that ICG-001 decreased the level of BRG1 protein expression in vitro. mTECs were stimulated with ICG-001 (5 µM) for 24 h. Representative Western blot of BRG1 (M) and quantitative data (N) are presented. **P<0.01 versus control groups (n=3). (O) Quantitative real-time polymerase chain reaction (qRT-PCR) showed that ICG-001 increased the mRNA level of BRG1 in vitro. mTECs were stimulated with ICG-001 (5 µM) for 24 h. *P<0.05 versus control groups (n=3). (P,Q) Western blot analysis showed that CQ recovered ICG-001-induced BRG1 protein down-regulation. mTECs were incubated with ICG-001 in the absence or presence of autophagy–lysosomal inhibitor CQ (20 μM) for 24 h. Representative Western blot of BRG1 (P) and quantitative data (Q) are presented. **P<0.01 versus control group; ##P<0.01 versus ICG-001 alone (n=3). Abbreviations: Baf A1, bafilomycin A1; CQ, chloroquine; RAPA, rapamycin.