PINCH1 blocks WT1-mediated podocalyxin expression in human podocytes.

<p><i>A</i>, RT-PCR analyses demonstrate that PINCH1 blocked WT1-stimulated podocalyxin mRNA expression in podocytes. Cells were transfected with expression vectors for PINCH1, WT1 or both, respectively. RT-PCR amplification of housekeeping GAPDH was performed in an identical manner to serve as controls. <i>B</i>, Graphic presentation shows the relative PINCH1 mRNA abundance in different groups after normalization with GAPDH. Data are presented as mean ± SEM of three independent experiments. *<i>P</i><0.05. <i>C</i>, Western blot analyses show that PINCH1 blocked WT1-mediated podocalyxin protein expression. Human podocytes were transfected with different plasmids as indicated for 48 h. Total cell lysates were immunoblotted with specific antibodies against podocalyxin and actin, respectively. <i>D</i> and <i>E</i>, Knockdown of PINCH1 in podocytes promotes podocalyxin expression. Human podocytes were transfected with either control or PINCH1-specific siRNA. The expression of PINCH1 (<i>D</i>) and podocalyxin (<i>E</i>) was assessed by quantitative RT-PCR. **<i>P</i><0.01 (n = 3). <i>F</i>, PINCH1 represses WT1-activated podocalyxin gene promoter activity. Human podocytes were co-transfected with different plasmids as indicated with luciferase- podocalyxin gene promoter reporter construct (pGL3-podocalyxin) for 48 h. Equal amounts of DNA were present in each transfection. Data are presented as mean ± SEM of three independent experiments. *<i>P</i><0.05.