Figure 3

Activation of DD1 leads to expression of FASTKD2 which mediates the apoptotic response. (A) LNCaP-AI cells stably expressing DD1-ERT2 or DD1(S28A)-ERT2 were incubated with 1 uM 4-OHT for 8 h and then analyzed for DD1 and DD1(S28A) expression by immunofluorescense using FLAG-M2 antibody (green) and apoptosis by TUNEL assay (red). Nuclei were stained with DAPI (blue). (B) LNCaP-AI cells stably expressing DD1-ERT2 were transfected with a Control siRNA or an siRNA known to knock down expression of FASTKD2 [5]. Twenty-four h later the cells were transfected again with the siRNAs as described in the Methods section. Twenty h after the second siRNA transfection cells were incubated with 4-OHT for 15 h. Cells were then fixed and permeablized for TUNEL assay (red). Nuclei are stained with DAPI (blue). Cells treated with the FASTKD2 siRNA exhibited essentially no apoptosis after 4-OHT incubation while cells treated with the Control siRNA showed extensive apoptosis.