Fig. 5

Smad2/3 mediates TGFβ-induced lnc-LFAR1 expression in HSCs. a, b Relative expression levels of Smad2, Smad3 and lnc-LFAR1 in primary HSCs infected with lenti-shSMAD2 or lenti-shSMAD3 or lenti-control virus were examined by qRT-PCR a and western blot b compared to GAPDH. Uncropped blots of this figure accompanied by the location of molecular weight markers are shown in Supplementary Fig. 18. c, d ChIP analyses of primary HSCs treated with or without 10 ng ml⁻¹ TGFβ for 24 h were conducted on lnc-LFAR1 (primer set a–d), PAI (the positive control; primer e) and GAPDH (the negative control; primer f) promoter regions using anti-Smad2/3 antibody. Enrichment was shown relative to input. e Diagram of the predicted three Smad2/3 binding sites in the lnc-LFAR1 promoter region. Points mutation of binding site 1 (Mut1), and binding sites 2 and 3 (Mut2) of Smad2/3 were indicated. f Luciferase analysis. Primary HSCs were transfected with the luciferase reporter constructs harboring either Smad2/3 binding sites or the mutated binding sites for 48 h, and further treated with 10 ng ml⁻¹ TGFβ for additional 24 h. The cells were lysed for dual luciferase analysis. The Renilla was transfected as an internal control. In a, d and f, the number of biological replicates for each experiment was n ⩾ 3. Data are presented as means ± s.e.m. P values were analyzed by Student’s t-test. *P < 0.05. *P < 0.05 vs shRNA-control in a; and *P < 0.05 vs Ctrl in d and f