Figure 2
From: RNA-binding motif protein 47 inhibits Nrf2 activity to suppress tumor growth in lung adenocarcinoma
Regulation of RBM47 by TGF-β and TTF-1 in lung adenocarcinoma cells. (a, b) After the 24-h treatment of A549 cells and H441 cells with 2.5 ng/ml TGF-β, expression levels of RBM47 mRNA and protein were examined by qRT–PCR (a) and immunoblot (b) analyses, respectively. α-tubulin was used as a loading control in (b). Protein expression was quantified using Image J and normalized to that of lane 1 and is indicated below each panel. (c) ChIP-seq data of Smad3 and TTF-1 binding at the RBM47 locus in H441 cells. An arrow indicates the transcription starting site of RBM47. TTF-1 binding (top panel) and Smad3 binding (center and bottom panels) in TGF-β-treated H441 cells transfected with control or TTF-1 siRNAs, respectively, are shown. (d) H441 cells transfected with control or TTF-1 siRNAs were examined by immunoblot analysis to evaluate the effect of TTF-1 on the expression of RBM47 protein. The arrow indicates the endogenous TTF-1 protein. (e) A549 cells were transfected with human RBM47 promoter-reporter construct in combination with adenoviral FLAG-TTF-1 or LacZ infection. At 48 h after infection, cells were harvested and assayed for luciferase activities. Averages and standard deviations of the two biological replicates were shown for each condition. Expression of FLAG-TTF-1 was confirmed by immunoblotting (bottom panel). (f) H441 cells were transfected with human RBM47 promoter-reporter construct in combination with control or TTF-1 siRNAs (siTTF-1#1 and siTTF-1#2). At 48 h after transfection, cells were harvested and assayed for luciferase activities. siCT: control siRNA. Averages and standard deviations of the two biological replicates were shown for each condition. *P<0.01. (g) A549 cells were transfected with human RBM47 promoter-reporter construct in combination with control or Smad3 siRNAs (siSmad3#1 and siSmad3#2). At 24 h after transfection, cells were treated with 2.5 ng/ml TGF-β for 24 h and harvested for luciferase activities. Averages and standard deviations of the two technical replicates were shown for each condition. *P<0.01.
