FIGURE 6.

SSeCKS facilitates PMA-induced CaP cell shape change. A, SSeCKS levels in CaP cells. IB for SSeCKS or β-actin from lysates of MLL cells +/− SSeCKS (2–6 cells grown, respectively, in regular or tetracycline-containing medium) versus EP12 cells (immortalized, untransformed rat prostate epithelial). Right panel, LNCaP or LNCaP-C4-2 cells were grown under pre- (−2 and −1) and confluent (2 and 3) conditions with the numbers representing days before or after confluence. B, SSeCKS increases PMA-induced cell rounding in MLL cells. Subconfluent cultures of MLL/2-6 cells grown in the absence or presence of tetracycline (+ and − SSeCKS (SS), respectively) treated with PMA (100 nm). ′, minutes. Error bars, S.E. from triplicate experiments. C, D, and E, SSeCKS prevents PMA-induced apoptosis and rescues PMA-induced cell rounding in LNCaP or LNCaP-C4-2 cells. Phase-contrast microscopy of subconfluent LNCaP cultures shows PMA-induced cell flattening (C). Cells were transfected transiently with GFP fusions of either the α or β SSeCKS isoform (24, 55), treated for 24 h with 100 nm PMA or DMSO vehicle, and then analyzed by FACS after staining for phycoerythrin-Annexin V (white columns) or 7-aminoactinomycin D (7-AAD) (black columns) and then gating for double staining with GFP-positive cells (D). Error bars, S.E. from triplicate experiments. Cells transfected with empty vector (pEGFP), FL SSeCKS-GFP or SSeCKS[Δ533–900]-GFP were analyzed by fluorescence microscopy for cell rounding (E). Error bars, S.E. from triplicate experiments. *, p < 0.002; **, p < 0.01.