Fig. 4: Smads and LXRα bind to the α-SMA gene promoter. | Oncogenesis

Fig. 4: Smads and LXRα bind to the α-SMA gene promoter.

From: LXRα limits TGFβ-dependent hepatocellular carcinoma associated fibroblast differentiation

Fig. 4

a Diagram of the hACTA2 gene promoter indicating the transcriptional start site (arrow, +1), putative Smad- (CAGA motif, red boxes) and LXR-binding sites (blue boxes) on the promoter, with numbering relative to the start site corresponding to the first base pair of each CAGA motif and the ends of the promoter fragment. Promoter fragments detected by ChIP (green) and used for DNAp (grey) are shown. b ChIP-qPCR analysis of endogenous Smad2/3 binding to three different fragments of the hACTA2 gene promoter as indicated in (a), or to the human PAI-1 gene, used as positive control. Mean ± SD values are plotted. Experiments were performed in biological duplicates (nb = 2), each of them in technical triplicate (nt = 3). Statistical comparison (two-sided t-test) indicates significant differences, ***p < 0.001. c DNA-precipitation (DNAp) assays using an hACTA2 gene promoter-specific oligonucleotide and human 293-T cell lysates after transient transfection with pCDNA3-Flag-Smad3 and pCMX-LXRα in the absence (−) or presence (+) of stimulation with 5 ng/ml TGFβ1 for 2 h followed by sequential immunoblotting for transfected Smad3 and LXRα. d DNAp using lysate from human 293-T cells transfected only with pCMX-LXRα and stimulated with 5 ng/ml TGFβ1 for 2 h and/or 5 µM T0901317 (T09) for 16 h followed by sequential immunoblotting for endogenous Smad3 and transfected LXRα. Densitometric quantification of the Smad3 and LXRα protein bands is shown. e DNAp using lysate from untransfected HepG2 cells stimulated with 5 ng/ml TGFβ1 for 2 h and/or 5 µM T0901317 (T09) for 18 h followed by sequential immunoblotting for endogenous Smad3 and endogenous LXRα. f DNAp using a multimerized Smad-binding element (CAGA) oligonucleotide as positive control followed by sequential immunoblotting for endogenous Smad3 and LXRα. Total cell lysates (TCL) indicate expression of each protein, relevant proteins are marked with an arrow, molecular size markers are indicated in kDa, and grey boxes with a plus sign mark control DNAp with streptavidin beads in the absence of specific oligonucleotide to indicate non-specific, background precipitation. Experiments were performed in biological duplicates (nb = 2), each of them in technical triplicate (nt = 3)

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