Fig. 2: Pharmacological inhibition of GSK3β by TDZD-8 or LiCl attenuates the TGF-β1 induced TEC profibrogenic plasticity in a dose-dependent fashion.

A The immortalized murine renal proximal tubular epithelial cells (TKPT) were treated with or without TGF-β1 (0.5, 1, 2, 4 ng/ml) for 24 h. Cell lysates were prepared for immunoblot analysis for GSK3β and GAPDH. Representative immunoblots were shown. B TKPT were treated with or without TGF-β1 (2 ng/ml) for 12 h, 24 h, or 48 h. Cell lysates were prepared for immunoblot analysis for GSK3β and GAPDH. Representative immunoblots were shown. C TKPT were treated with TGF-β1 (2 ng/ml) for 24 h following pretreatment with different dose of TDZD-8 (0, 2, 5, 10 μmol/L) or (D) LiCl (0, 2, 5, 10 mmol/L) for 30 min. Cell lysates were prepared for immunoblot analysis for E-cadherin, vimentin, fibronectin (FN), PAI-1, CTGF, pH3, and GAPDH. Representative immunoblots were shown. E, F Densitometric analyses of the expression of E-cadherin, vimentin, FN, PAI-1, CTGF, and pH3, as normalized to the expression of GAPDH based on immunoblot analysis. ^*P < 0.05 versus TGF-β1 treatment group (n = 3, t-test). G TKPT were treated with TGF-β1 in the presence or absence of LiCl (10 mmol/L) for 24 h and then fixed for immunofluorescent staining of E-cadherin, ZO-1, vimentin, pH3, and FN with nuclear counterstaining with DAPI. Representative fluorescent micrographs were shown. Scale bar = 100 μm. CTGF, connective tissue growth factor; DAPI, 4′,6-diamidino-2-phenylindole; FN, fibronectin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PAI-1, plasminogen activator inhibition-1; pH3, phosphorylated histone H3; ZO-1, zonula occludens-1.