Figure 4

INPP4B regulation of IL-8 and PAK6 is independent of Akt. PC-3 Tet-On #14 cells were cultured for 2 days with or without doxycycline on matrigel coated plates in full medium. (A) INPP4B induction levels were determined by quantitative RT-PCR. (B) Validation of gene expression in samples from (A): IL-8, COL6A3, HAS2 and PAK6. (C) Negative control, #4, and #14 PC-3 cell lines were treated as in (A) and cellular extracts were analyzed for FLAG-INPP4B, PAK6, and tubulin expression. (D) Negative control, #4, and #14 clines were induced with 0.5 μg/ml of doxycycline for 48 hours in complete medium. Conditioned medium was collected, cleared by centrifugation and concentrations of secreted IL-8 protein were determined using human IL-8-specific capture ELISA kit. Values were calculated as a percent of IL8 expression in negative control PC-3 clone. (E-F) PC-3 cells cultured in growth medium were treated with DMSO (vehicle), 0.5 μM AZD5363, 5 μM AZD5363 or 10 μM LY294002 to inhibit Akt or PI3K respectively. RNA was extracted and analyzed for expression of IL-8 (E) and PAK6 (F) by quantitative PCR and normalized to 18S. (G) Inhibition of PI3K and Akt by LY294002 and AZD5363 was confirmed by analyzing expression and activation status of the ribosomal protein S6 by Western blot analysis of PC-3 clone #14 cells treated with 0.5 or 5 μM AZD5363 (AZD) and 10 μM LY294002 (LY). Tubulin was used as a loading control. Data are presented as means ± SEM. * P < 0.05, **P < 0.01, *** P < 0.001.