Figure 5. IGF-I induces Survivin through a Smad dependent mechanism.

(A) NRP-152 cells plated overnight in GM3 were treated at various times with LR³-IGF-I for up to 72 h, and cell lysates were analyzed for Western blot expression of Survivin, and P-Smads 2 and 3. (B) NRP-152 cells stably expressing sh-Smads 2, 3, and 2+3 or lentiviral sh-LacZ (control) were treated with 2 nM LR³-IGF-I or vehicle for 24 h prior to Western blot analysis for Survivin and Smads 2 and 3. (C) NRP-152 cells stably expressing sh-Smads 2+3 or lentiviral sh-LacZ (control) were treated with DMSO vehicle or 10 µM SB431542 for 2 h prior to treatment with 2 nM LR³-IGF-I or vehicle for 24 h, and changes in Survivin expression was assessed by Western blot analysis. (D) NRP-152 cells were treated with either 10 µM HTS466284 or 10 µM SB43152 for 2 h prior to treatment with 2 nM LR³-IGF-I or vehicle for 24 h, and changes in Survivin expression were assessed by Western blot analysis. (E,F) RWPE-1 and VCaP cells plated in GM3 were treated with LR³-IGF-I and the TGF-β receptor kinase inhibitors HTS466284 (HTS) or TKDI for 24 h prior to lysing cells for Western blot analysis of Survivin expression. Results are representative of two to three separate experiments.