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. Author manuscript; available in PMC: 2018 Apr 24.
Published in final edited form as: Biochem J. 2017 Apr 24;474(9):1531–1546. doi: 10.1042/BCJ20160943

Figure 1. TGF-β-SMAD signaling downregulates PPARγ at the transcriptional level.

Figure 1

(A, B) IMR-90 human lung fibroblasts were transfected with SMAD3 or SMAD4-targeted siRNA, or with scrambled siRNA as control, and then treated with or without TGF-β (2 ng/ml). Following treatment: (A) PPARG mRNA was measured by real-time PCR, and (B) PPARγ, SMAD3 and SMAD4 protein expression was assessed by Western blotting. (C) IMR-90 fibroblasts were treated with or without TGF-β (2 ng/ml) and nascent mRNA was captured by Click-iT Nascent RNA Capture kit, with relative mRNA levels of PPARG and ACTA2 being measured by real-time PCR. (D) Nuclei isolated from IMR-90 lung fibroblasts were incubated with biotin-16-UTP and a nuclear run-on assay was performed. Labelled, newly synthesized transcripts were collected using streptavidin-conjugated magnet beads and relative levels of PPARG and ACTA2 mRNA were determined by real-time PCR. (E) mRNA stability was determined by incubation with growth medium containing 5-EU for 24 h followed by incubation with unlabeled growth medium for the indicated periods. Total mRNA was then isolated and labelled mRNA captured and analyzed with Click-iT Nascent RNA Capture Kit as described in Experimental Procedures. The data are expressed as the mean ± SD with n = 3–4 and the results were reproduced in two to three independent experiments; **P < 0.01, ***P < 0.001, n.s. = non-significant.