Figure 5.

Upstream Mechanism of lncITPF

(A) TGF-β1 increased the lncITPF promoter activity. Cells transfected with pGL3-lncITPF vectors displayed significantly higher luciferase activity than empty pGL3. Similarly, cells treated with TGF-β1 displayed significantly higher luciferase activity than those without. Cells were transfected with pGL3-lncITPF vectors or an empty vector with or without TGF-β1 treatment for 48 and 72 hr. Luciferase activity was normalized to renilla. (B) Result of Cas9 technology showed that lncITPF had its own promoter located inside the Itgbl1 gene, and they could not be co-transcribed. (C) Signal pathway inhibitors were used to detect the change in lncITPF expression. Only the inhibitor SB431542 of the smad2/3 pathway blocked the expression of lncITPF. (D) ChIP-qPCR analysis of the binding of smad2/3 at the lncITPF promoter region with or without TGF-β1 treatment. ChIP-purified DNA targeting of the indicated genes was analyzed by qPCR. L1 and L2 indicated the different sequences of lncITPF. (1) MRC-5 input; (2) MRC-5 chip DNA; (3) MRC-5 input treated with TGF-β1; (4) MRC-5 chip DNA treated with TGF-β1. (E) The ChIP-qPCR gel images were quantified and analyzed. Each bar represents the mean ± SD; n = 6; *p < 0.05, **p < 0.01, and ***p < 0.001.