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. 2018 Aug 28;293(42):16364–16375. doi: 10.1074/jbc.RA118.004073

Figure 2.

Figure 2.

Yap/Taz mediates Rictor/mTORC2-stimulated fibroblast activation. A, Western blot assay showing that ablation of Rictor reduced TGFβ1-induced FN and α-SMA expression in kidney fibroblasts. B, Western blot assay (left) and quantitative analysis (right) showing the protein abundance for FN and α-SMA in Rictor+/+ and Rictor−/− primary cultured kidney fibroblasts. Fibroblasts were treated with TGFβ1 for 24 h. *, p < 0.05 compared with Rictor+/+ fibroblasts (n = 4). GAPDH was probed to show the equal loading of the samples. C, Western blot analysis showing that PP242 could dose-dependently inhibit TGFβ1-induced FN and α-SMA expression in NRK-49F cells. D, immune staining showing that PP242 could inhibit TGFβ1-induced NRK-49F cell activation. Cells were co-stained with 4′,6-diamidino-2-phenylindole (DAPI) to visualize the nuclei. Scale bar, 5 μm. E, Western blot analyses showing the abundance of FN and α-SMA expression (left) and p-Smad3 (right). NRK-49F cells were treated with verteporfin, followed by TGFβ1 administration for 24 h (left) or 30 min (right). F, representative micrographs showing that verteporfin could abolish TGFβ1-induced fibroblast activation in NRK-49F cells. Cells were co-stained with DAPI to visualize the nuclei. Scale bar, 5 μm. G and I, Western blot analyses demonstrating the down-regulation of Yap (G) or Taz (I) after siRNA transfection, respectively. H and J, NRK-49F cells were pretreated with scramble, Yap, or Taz siRNA for 24 h, followed by TGFβ1 treatment for 24 h. Western blot analysis shows that knocking down Yap or Taz could reduce TGFβ1-induced FN and α-SMA expression (left). Right, graphic presentation of FN and α-SMA protein abundance in NRK-49F cells. *, p < 0.05 compared with control (n = 3); #, p < 0.05 compared with TGFβ1-treated cells (n = 3). K and L, Western blot assay showing the expression of exogenous Taz (S89A) in NRK-49F cells after Taz-S89A plasmid transfection (K). Western blot assays show the abundance for FN and α-SMA in NRK-49F cells (L). NRK-49F cells were transiently transfected with pTaz-S89A, followed 24 h later by TGFβ1 with or without PP242 treatment for 24 h. M, representative micrographs showing the immunostaining for FN and α-SMA in fibroblasts after various treatments as indicated. Cells were co-stained with DAPI to visualize the nuclei. Scale bar, 5 μm. Error bars, S.E.