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. 2013 Feb 20;288(16):11047–11065. doi: 10.1074/jbc.M112.438473

FIGURE 4.

FIGURE 4.

Lack of influence of ELK1-dependent androgen/AR signaling on cell survival, ELK1 phosphorylation, or regulation of immediate early genes. A, hormone-depleted LNCaP cells were infected with ELK1 shRNA 1, ELK1 shRNA 2, or control (Ctrl) shRNA lentivirus. 96 h later, cells were treated with vehicle (Veh) or R1881 (1 nm) for 48 h. Apoptosis was measured by the Annexin V assay. As a positive control for the assay, apoptosis was induced by treatment with cisplatin (100 μm) for 24 and 48 h. B, hormone-depleted C4-2 cells were infected with ELK1 shRNA 1, ELK1 shRNA 2, or control shRNA lentivirus. 6 days later, apoptosis was measured by the Annexin V assay. As a positive control for the assay, apoptosis was induced by treatment with cisplatin (100 μm) for 24 and 48 h. C, LNCaP cells were treated with vehicle or R1881 (1 nm) for 48 h and harvested for RNA and protein. ELK1 expression was measured using quantitative real time PCR, and the protein lysates were analyzed by Western blot for ELK1 with GAPDH as loading control (inset). D, serum-starved (Ser starv) LNCaP cells were treated with vehicle or phorbol 12-myristate 13-acetate (PMA) (10 μm) for 1 h. LNCaP cells plated in hormone-depleted medium were treated with vehicle or R1881 (1 nm) for the indicated times. Cell lysates from all the samples were analyzed by Western blot for phospho-ELK1 (P-Elk1) or ELK1 protein with GAPDH as the loading control. E, serum-starved LNCaP cells were stimulated with 20% FBS for the indicated times. LNCaP cells plated in hormone-depleted medium in charcoal-stripped serum (CS SERUM) were treated with vehicle or R1881 (1 nm) for the indicated times. RNA was harvested, and the induction of endogenous c-FOS, EGR1, PSA, TMPRSS2, CDC6, and DTL by serum or by R1881 was measured by quantitative real time PCR. For serum stimulation, mRNA levels are plotted relative to the values for serum starvation. For R1881 treatment, the mRNA levels are plotted relative to their respective vehicle controls. R denotes R1881. * and **, p < 0.001. Error bars represent S.D.