Role of Heparan Sulfate 2-O-Sulfotransferase in Prostate Cancer Cell Proliferation, Invasion, and Growth Factor Signaling
Figure 6
Inhibition of p38 MAPK or ATF2 results in decrease in 2OST mRNA. (a) Schematic of 2OST promoter with regions of predicted ATF2 binding sites (A1 and A2). (b) Real-time PCR analysis of 2OST levels in cells treated with either DMSO (control, black bars), 40 μM (gray bars), or 80 uM SB202190 (specific p38 MAPK inhibitor, white bars) for 12 hours. Error bars indicate standard deviation. (c) 2OST promoter β-galactosidase reporter assay in cells treated with either DMSO control (black bars) or 80 μM SB202190 (gray bars). Full-length 2OST promoter represents region 2500 bases upstream to 500 bases downstream of transcription start site. 4C represents region 1500 bases upstream to 500 bases downstream from start site. Error bars indicate standard deviation. (d) Western blot showing decreased levels of phospho-ATF2 in cells treated with 80 μM SB202190. (e) Inhibition of ATF2 by siRNA results in decreased levels of 2OST mRNA. Real-time PCR analysis of ATF2 and 2OST normalized to levels of 18S. Black bars represent samples treated with scrambled siRNA, gray bars represent either ATF2 or 2OST levels in ATF2 siRNA-treated cells. Error bars indicate standard deviation. Asterisk indicates . (f) Chromatin Immunoprecipitation analysis of C4-2B total chromatin, ATF2 IP, no antibody (NoAB), and Mock samples. Samples were analyzed by PCR with primers flanking each predicted ATF2 site. Positive control primers were used from the human insulin promoter [36].
