Figure 12.

GATA-6 (GATA6) silencing affects caveolin-1 (CAV1) gene expression, transcriptional activity, and promoter occupancy in primary human BSM cells. A: RT-PCR analysis of GATA-6 and caveolin-1 in scrambled and GATA-6 shRNA-transfected human BSM cells. GAPDH was amplified as control. B: Densitometric analysis of the bands in A for GATA-6 and caveolin mRNA is presented relative to GAPDH. C: Immunoblot analysis of total protein from cells transduced with scrambled and GATA-6 shRNA lentivirus vectors. GAPDH was probed as a loading control. D: Densitometric analysis of the bands in C for GATA-6 and caveolin-1 protein relative to GAPDH. E: ChIP analysis of the CAV1 promoter with GATA-6 antibody in human BSM cells transduced with scrambled and GATA-6 shRNA. GATA-6 binding motif on human CAV1 promoter was amplified at PCR using the primers given in Table 1. Preimmune serum was used as negative control. F: Quantitation of the band intensity from E is expressed as percentage of input DNA. G: Relative luciferase activity in human BSM cells transfected with scrambled (black bars) and GATA-6 (gray bars) siRNA. Reporter firefly luciferase activities were normalized to Renilla luciferase activity. In B, D, F, and G data are given as mean (SD) of three independent experiments.