FIGURE 2

P₃-25 inhibits expression of ICAM1 and Cox-2. (a and b) Jkt/TNF, HuT-78, HT-29, DU145, and PC3 cells were treated with P₃-25 (100 nM) for different times at 37°C. Jkt/TNF and HuT-78 cells were separately treated with 5  M BAY 11-7082 (designated as BAY) for 12 h. ICAM1 (a) or Cox-2 (b) was detected from whole-cell extract proteins (100 g) by Western blot. Tubulin was detected by reprobing both blots. (c) All these cells were cotransfected with vector, Cox-2-Luciferase, and GFP constructs using SuperFect reagent for 3 h. Cells were washed, cultured for 12 h, and counted for GFP-positive cells. Luciferase activity was assayed from whole-cell extracts prepared from P₃-25- (for different times) or BAY- (for 12 h) treated cells, considering vector alone value as one fold. (d and e) HuT-78 or DU145 cells were treated with 100 nM P₃-25 for different times or 5  M BAY for 12 h. Total RNA was isolated and Cox-2, ICAM1, and actin were detected by RT-PCR followed by PCR using Cox-2, ICAM1, and actin-specific primers at 305, 447, and 496 bp bands, respectively