Figure 4.

NF-ĸB is a mediator of 5-Aza-2′deoxycytidine-induced HEXIM1 expression. (A) LNCaP and C4-2 cells were treated with 5-AzadC. Cell lysates were prepared and expression of phospho-NF-ĸB and NF-ĸB were analyzed by western blots. Represented are blots cut into strips prior to blotting to minimize the amounts of antibodies required. Figures are representative of at least 3 independent experiments. *P < 0.05 vs. Control. (B) C4-2 and LNCaP cells were treated with 2 µM of VE-822 or caffeine for 2 h, followed by 5-AzadC (5 µM) for 2 h. Cells were processed for ChIP analyses of the occupancy of NF-ĸB on the HEXIM1 promoter. Input DNA was used as normalization control. IgG control was used as a negative control. *P < 0.05 and **P < 0.01 vs. Control. (C) C4-2 and LNCaP cells were infected with lentiviruses expressing control or NF-ĸB shRNA, and selected with puromycin. Some cells were treated with 2 µM of VE-822 or caffeine for 2 h, followed by 5-AzadC (5 µM) for 2 h. Cell lysates were prepared and expression of indicated proteins were analyzed by western blot. Represented are blots cut into strips prior to blotting to minimize the amounts of antibodies required. Figures are representative of at least 3 independent experiments. (D) LNCaP and C4-2 cells were infected with control or NF-ĸB shRNA lentiviruses and selected with puromycin. Some cells were treated with 5-AzadC (5 μΜ, 2 h). Cells were then processed for ChIP analyses of recruitment of CDK9 and NF-ĸB to the HEXIM1 promoter. Input DNA was used as normalization control. IgG control was used as a negative control. Figures are representative of at least 3 independent experiments. *P < 0.05 and **P < 0.01 vs. Control.