Fig. 3: Inhibition of ATF3 reduced TGF-β2 expression and Sema7A-induced EndMT.

a ATF3 mRNA level was analyzed by qPCR normalized to GAPDH. Fold changes are shown. Data are mean ± SEM, N = 3, ***p < 0.001 b ATF3 protein expression were analyzed by western blotting, normalized to tubulin. Data are mean ± SEM, N = 3, ***p < 0.001. c TGF-β2 mRNA level was analyzed by qPCR normalized to GAPDH. Fold changes are shown. Data are mean ± SEM, N = 3, *p < 0.05; **p < 0.01. d The concentration of TGF-β2 in cell supernatant was detected by ELISA among Con335-HUVECs, Sema7A-HUVECs, and Sema7A-HUVECs + ATF3-siRNA. N = 10. Data are mean ± SEM, *p < 0.05; ***p < 0.001. e Chip-qPCR product in agarose gel electrophoresis. f Chip-qPCR TGF-β2 percentage of input in con335-HUVECs and Sema7A-HUVECs were analyzed by qPCR normalized to IgG. Data are mean ± SEM, N = 3, **p < 0.01. g Schematic graph of the constructed reporter plasmid. TGF-β2 mut indicates the TGF-β2 mutation promoter region in ATF3 binding site. The mutated nucleotides in TGF-β2 fragments are in red letters. h Luciferase reporter assays were performed on HEK 293 T cells. Data are mean ± SEM, N = 3, **p < 0.01 vs negative control. i, j ATF3-overexpression plasmid was transfected to HUVECs, and the mRNA levels of TGF-β2 and TGF-β1 were performed by qPCR normalized to GAPDH. Fold changes are shown. Data are mean ± SEM, N = 3, **p < 0.01. k, l P-Smad3 protein in cells treated with siRNA was analyzed by Western blotting, normalized to total Smad3. Data are mean ± SEM, N = 3, *p < 0.05; **p < 0.01. m–q CD31 and α-SMA RNA and proteins in cells treated with siRNA or control was analyzed by qPCR and western blotting. Data are mean ± SEM, N = 3, *p < 0.05; **p < 0.01; ***p < 0.001.