Figure 3.

AR RNAi inhibits PMA induction of TNFα and TRAIL. LNCaP cells growing in normal medium were transfected with either an RNAi duplex for AR or a control (C) duplex using the Amaxa Nucleofector, and 48 h later treated for 1 h with either 100 nM PMA or vehicle. For mRNA determinations, RNA was extracted 3 h after treatment. For cytokine determinations, CM was collected 24 h after treatment. Panel A: Representative Western blot showing AR depletion and PKCδ down-regulation in AR-depleted cells 48 h after RNAi transfection. Expression levels, relative to control RNAi, have been determined by densitometry and are shown below each corresponding Western blot. Panel B: Apoptotic effect of CM-PMA from LNCaP cells collected from cells subjected to AR or control RNAi. The percentage of apoptotic cells was determined by DAPI staining 24 h after addition of CM-PMA. Panel C: TNFα and TRAIL mRNA levels were determined by real-time PCR 3 h after PMA treatment in LNCaP cells subject to either AR or control RNAi. Results are normalized to endogenous GAPDH mRNA levels and expressed as fold-increase relative to those in control cells treated with vehicle. Panel D: TNFα and TRAIL levels in CM-Veh or CM-PMA collected from LNCaP cells subjected to AR or control RNAi, as determined by ELISA. In all cases, a representative experiment is shown and results are presented as mean ± SD of triplicate samples. Similar results were obtained in two additional experiments. CM-PMA, conditioned medium from PMA-treated cells; CM-Veh, conditioned medium from vehicle-treated cells.