Fig. 2: The expression of Ets-1 was directly regulated by TGF-β1-Smad2/3. | Cell Death & Disease

Fig. 2: The expression of Ets-1 was directly regulated by TGF-β1-Smad2/3.

From: Ets-1 deficiency alleviates nonalcoholic steatohepatitis via weakening TGF-β1 signaling-mediated hepatocyte apoptosis

Fig. 2

a–d Primary hepatocytes from WT mice were (a, b) treated with different concentrations of TGF-β1 for 24 h or (c, d) harvested at different time points under the treatment of 10 ng/mL TGF-β1. a, c The relative mRNA level of Ets-1 was measured using qPCR. b, d Immunoblots of Ets-1 are shown. e, f WT primary hepatocytes were pretreated with SB-431542 (10 μM) for 12 h. Then, 10 ng/mL TGF-β1 was added to the cells for 24 h. e The relative expression of Ets-1 was measured using qPCR. f Immunoblots of Ets-1. g, h siRNAs of the negative control (siNC) and Smad2/3 (siSmad2/3-1 and siSmad2/3-2) were added to primary hepatocytes for 36 h, before incubation with 10 ng/mL TGF-β1 for 12 h (g) or 24 h (h). g The expression of Smad2, Smad3, and Ets-1 were examined. h Immunoblotting for Smad3, Smad2, and Ets-1 was performed. i The promotor of Ets-1 between –614 bp and –3 bp is shown. The locations of Sequence-1 and Sequence-2 are marked by a red box. The sequences of (C/AGAC) were Smad2/3-specific DNA-binding elements. a, b The ChIP assay of Ets-1 promoter used primary hepatocytes treated with TGF-β1 (10 ng/mL) for 6 h. An anti-Smad2/3 polyclonal antibody was used for precipitation. PCR analysis of the input and immunoprecipitation with IgG and an anti-Smad2/3 antibody were performed. Quantitative data are expressed as mean ± SEM (at least three independent experiments). *P < 0.05 and **P < 0.01

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