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. 2017 Dec 13;8(12):3214. doi: 10.1038/s41419-017-0050-3

Fig. 7. Regulation of PNPO expression by the TGF-β signalling pathway.

Fig. 7

a, b, c Effect of TGF-β1 on PNPO expression. SK-OV-3 and OVCAR-3 cells were treated with TGF-β1 at different concentrations (0, 1, 10 ng/ml). a PNPO mRNA expression was detected after 24 h treatment. b PNPO protein expression was detected after 48 h treatment. c Semi-quantitative analysis of the relative optical density of protein bands in b (n = 3). d, e, f Effect of TGF-β type I receptor kinase inhibitor SB431542. Cells were pre-treated with 10 µm SB431542 for 30 min and then treated with 1 ng/ml of TGF-β1. d PNPO mRNA expression was detected after TGF-β1 treatment for 24 h. e PNPO protein expression was detected after TGF-β1 treatment for 48 h. f Semi-quantitative analysis of the relative optical density of protein bands in e (SK-OV-3, n = 5; OVCAR-3, n = 4). Phospho-Smad2 (p-Smad2) and β-actin were used as controls in western blot analysis. g A schematic diagram illustrated the sequences of the putative miR-143-3p binding site in the PNPO 3′-UTR and its mutant. h Dual-luciferase reporter assay. The interaction of miR-143-3p with the PNPO mRNA at 3′-UTR in HEK293T cells was confirmed (n = 3). i Effect of miR-143 mimics (50 nm for 72 h) on PNPO protein expression detected by western blot in OVCAR-3 cells. j Semi-quantitative analysis of the relative optical density of protein bands in I (n = 3). k Effect of miR-143 inhibitor (anti-miR-143) (150 nm for 72 h) on PNPO protein expression detected by western blot in OVCAR-3 cells. l Effect of TGF-β1 (0, 1 and 10 ng/ml for 3 h) on primary miR-143 (Pri-miR-143) expression detected by qRT-PCR in OVCAR-3 cells. m Effect of TGF-β1 (0, 1 and 10 ng/ml for 3 h) on mature miR-143 expression detected by qRT-PCR in OVCAR-3 cells. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01