Fig. 6

NFKBIA is a critical mediator of death induced by combinatorial AR targeting. LNCaP cells (1 × 10⁵ cells per well in 12-well plates) or VCaP cells (2 × 10⁵ cells per well in 12-well plates) were transfected in triplicate wells with 10 nM of either non-specific, scrambled siRNA [N.S.] or specific siRNAs [siRNA #1 and siRNA #4] targeting NFKBIA. LNCaP cells were transfected in triplicate wells with 1.5 μg of plasmid expressing either GFP or NFKBIA+GFP, for 4 h. The transfection medium was then overlaid with vehicle control [VEH], or the combination [BIC + VOR] to give a final concentration of 2.5 μM bicalutamide and 1 μM vorinostat in either PRF-RPMI 1640 supplemented with 10 % FBS and 1 % l-glutamine (LNCaP) or DMEM supplemented with non-essential amino acids, 1 % l-glutamine and 10 % FBS (VCaP). a Western blot analysis of lysates from LNCaP and VCaP cells transfected with either non-specific [N.S.] or specific [#1 and #4] NFKBIA siRNA. Steady state levels of IκBα at day three (LNCaP) or day six (VCaP) of treatment are shown, with hsp90 as a loading control. b Cells were counted at 3 days (LNCaP) or 6 days (VCaP) of treatment, and assessed for viability using trypan blue dye exclusion. Cell death is expressed as a percentage of total cell number. Values indicated are the mean of triplicate wells ± SEM, and are representative of three independent experiments. c Western blot analysis of lysates from LNCaP cells transfected with plasmids expressing GFP only [GFP] or NFKBIA and GFP [NFKBIA], and then treated with vehicle control [VC] or combination [BIC+VOR]. Steady state levels of IκBα at day three of treatment are shown, with hsp90 as a loading control. d Cells were counted at 3 days of treatment, and assessed for viability using trypan blue dye exclusion. Cell death is expressed as a percentage of total cell number. Values indicated are the mean of triplicate wells ± SEM, and are representative of three independent experiments. * = p < 0.05 using t-test