Fig. 10.

Effects of FFA4 knockdown on inhibitory effects of EPA and TUG-891 on cell proliferation. (A) DU145 cells were incubated with scrambled siRNA (negative control) or FFA4 siRNA as described in Materials and Methods. Whole-cell extracts were immunoblotted for FFA4 and actin (loading control). The percent knockdown (KD) was calculated after quantification by densitometry, and normalization to actin. (B and C) DU145 cells (B) and PC-3 cells (C) were incubated with scrambled siRNA (Scr) or FFA4 siRNA (F4i), or without siRNA (control), as described in Materials and Methods. Simultaneously, cells were treated with and without 20 µM EPA or 1 µM TUG-891, in the continued presence of 10% serum. (D–G) DU145 cells (D and E) and PC-3 cells (F and G) were treated as in (B) and (C), except that serum was removed for 24 hours prior to the addition of scrambled or FFA4 siRNA, without or without 20 µM EPA, 1 µM TUG-891, 10 µM LPA, 10 nM EGF, or vehicle as indicated. For (B)–(G), data points represent the mean ± S.E.M. of values from duplicate experiments, each conducted in duplicate (n = 4). *Values that were significantly different (two-way ANOVA followed by Tukey’s multiple comparisons test; **P < 0.01; ****P < 0.0001) from the untreated control, as well as from the scrambled siRNA control, for the time point tested. UT, untransfected.