Figure 5
Effect of inhibiting TGF-β signaling pathway on endoplasmic reticulum (ER) stress mediated cell death. The effect of inhibiting TGF-β signaling pathway on extracellular matrix (ECM) production was evaluated. iFECD was cultured with SB431542 (TGF-βR1 inhibitor) or Smad inhibitor, or knockdown TGF-βR1 by siRNA. Phosphorylation of Smad3 was suppressed by SB431542, knockdown TGF-βR1, and Smad inhibitor. Production of fibronectin by iFECD was evaluated by western blotting. Experiments were performed in duplicate. (b) TGF-β signaling pathway was blocked by SB431542 (TGF-βR1 inhibitor) or Smad inhibitor, or knockdown TGF-βR1 by siRNA. The effect of TGF-β signaling pathway inhibition on unfolded protein was evaluated by aggresome staining. Experiments were performed in triplicate. Scale bar: 50 µm. (c) Effect of inhibition of the TGF-β signaling pathway on endoplasmic reticulum (ER) stress. Expression of GRP78, ATF6 pIRE1α, and CHOP, and phosphorylation of IRE1α and PERK was evaluated by western blotting. GAPDH was used as internal control. Experiments were performed in triplicate. (d,e) Effect of inhibiting TGF-β signaling pathway on mitochondrial membrane potential (MMP) depolarization was evaluated by fluorescent microscope after JC-1 staining and flow cytometry. Nuclei were stained with DAPI for fluorescence microscopy analysis. CCCP was used as positive control to induce MMP depolarization. Experiments were performed in duplicate. Scale bar: 50 µm. The statistical analysis was performed by Student’s t-test. *P < 0.01. (f–h) Western blotting was preformed to evaluate the effect of inhibiting TGF-β signaling pathway on cleavage caspase 3 and PARP. Full-length blots are presented in Supplementary Fig. 1. Densitometry analysis of three independent experiments was performed with ImageJ and plotted as graphs. The statistical analysis was performed with the Student’s t-test. *P < 0.01.
