Figure 9.

TGF-β1 mediated fibrotic gene induction requires TAZ signaling and SMAD3-TAZ cooperation. A–F) Serum-deprived HK-2 cells at similar density, stably expressing confluent control (Con) shRNA or TAZ shRNA, were stimulated with TGF-β1 (2 ng/ml) for 24 h, and lysate extracts were immunoblotted for TAZ (A, B), CTGF (A, C), fibronectin (A, D), PAI-1 (A, E), pSMAD3 (A, F), vimentin, YAP, and GAPDH (A) expression. B–F) Summary of relative TAZ, CTGF, fibronectin, PAI-1, and pSMAD3 protein levels for 3 independent experiments, setting expression levels in the untreated controls shRNA to 1 in each case. G) HK-2 cells, stably expressing TAZ shRNA and infected with either CMV-TAZ– or vector CMV-control–expressing lentiviral particles, remained unstimulated or treated with TGF-β1 for 24 h. Cell extracts were processed by immunoblotting for TAZ, fibronectin, CTGF, and GAPDH expression. H) HK-2 cells were incubated with the YAP/TAZ inhibitor verteporfin for 18 h at the indicated doses before addition of TGF-β1 at 24 h. Lysate extracts were analyzed by Western blot for expression of CTGF, fibronectin, PAI-1, and GAPDH. I) Nuclear extracts of untreated control or TGF-β1-stimulated HK-2 cells were immune blotted for TAZ, and lamin A/C (a marker of nuclear loading) expression. J) Serum-deprived HK-2 cultures expressing SMAD3- or Control shRNA were stimulated with TGF-β1 for 24 h before immune blot analysis for SMAD3, PAI-1, fibronectin, and GAPDH expression. A representative blot is shown. Data in all histograms are means ± sd. NS, not significant. *P < 0.05, **P < 0.01.