phosphate transporter inhibitor inhibits endothelial to mesenchymal transition through restored RASAL1 expression.

<p>Cells were treated with Pi 3mM for 72 hours. (A) MeDIP result showing the demethylated promoter of RASAL1 by using PFA, correlated with restored mRNA expression of RASAL1. (B) qPCR analysis showed the restored RASAL1 mRNA expression levels by using phosphate transporter inhibitor (PFA). Results were normalized to reference gene GAPDH (expression is presented as means ± s.d., n = 3 independent experiments, **P<0.01). (C) ELISA assay indicating the normalization of total Ras with combination of PFA or RAS inhibitor farnesylthiosalicylic acid (FTS). (D) bright-field images showing the morphology change of HCAEC cells cultured under normal control condition, high Pi conditions, and Pi combined with FTS or PFA. Scale bars 25 μm. (E) Western blot analysis showing the expression of endothelial cell marker CD31 and fibroblast cell marker S100A4 in HCAEC cells exposed to normal control condition, high Pi conditions, and Pi combined with FTS or PFA (F) qRT-PCR data showing the mRNA expression levels of EndMT transcriptional factors (SNAIL, SLUG, and TWIST) and FSP1 in HCAEC cells under four conditions indicated above. Results were normalized to reference gene GAPDH (expression is presented as means ± s.d., n = 3 independent experiments, ***P<0.001).