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. 2021 Aug 6;135(15):1873–1895. doi: 10.1042/CS20210447

Figure 7. BRG1 mediates tubular senescence and fibrotic responses via Wnt/β-catenin pathway.

Figure 7

(A) BRG1 overexpression enhanced the Wnt/β-catenin-mediated gene transcription activity. mTECs and HEK-293T cells were co-transfected with TOPFlash reporter plasmid, Renilla luciferase reporter vector (an internal control to normalise the transfection efficiency) and BRG1 expression vector (pReceiver-M14-BRG1) or control vector (pReceiver-M14) for 48 h as indicated. ***P<0.001 versus pReceiver-M14 group (n=3). (B,C) Cytoplasmic and nuclear proteins in mTECs were isolated and Western blot analysis was performed to verify the nuclear translocation of β-catenin. mTECs were transfected with BRG1 expression vector (pReceiver-M14-BRG1) or control vector (pReceiver-M14) for 48 h. Representative Western blot (B) and quantitative data on the relative abundance of cytoplasmic and nuclear β-catenin protein (C) in two groups are presented. β-Actin and Histone-H3 were used as internal controls for the cytosolic or nuclear fraction, respectively. ***P<0.001 versus pReceiver-M14 group (n=4). (D) Immunofluorescence staining demonstrated that the expression and nuclear translocation of β-catenin were induced by BRG1 overexpression in mTECs. Scale bar, 10 μm. (E–I) Western blot analysis showed that overexpression of BRG1 increased the expression of active-β-catenin, MMP-7, snail-1, and PAI-1. In mTECs, representative Western blot (E) and quantitative data on the relative abundance of active-β-catenin (F), MMP-7 (G), snail-1 (H) and PAI-1 (I) proteins in two groups are presented. *P<0.05, **P<0.01 versus pReceiver-M14 group (n=4). (J–N) Western blots analysis showed that BRG1-induced p16INK4a, p21, p19ARF, and TGF-β1 expression were blocked by pharmacologic inhibition of Wnt/β-catenin pathway in vitro. mTECs were transfected with pReceiver-M14-BRG1, followed by stimulation with Wnt/β-catenin signalling inhibitor ICG-001 (5 µM) for 48 h. Representative Western blot (J) and quantitative data on the relative abundance of p16INK4a (K), p21 (L), p19ARF, (M) and TGF-β1 (N) proteins are presented. *P<0.05,**P<0.01 versus control group; ##P<0.01, ###P<0.001 versus pReceiver-M14-BRG1 alone (n=3).