Research Paper Volume 8, Issue 5 pp 958—973

Figure 2. FS-115 efficiently suppresses p70S6K1 activity in MDA-MB-231 breast cancer cell line. (A) Western blot analysis of MDA-MB-231 breast cancer cells, serum starved (time 0) and then stimulated for 20 minutes with wound fluids (WF) or serum starved, pre-treated 30 minutes with FS-115 or PF-4708671 (50-10-5-1μM, as indicated) then stimulated for 20 minutes with WF, always in the presence of the inhibitor. p70S6K, AKT, MAPK and mTOR signaling pathways were analyzed. Vinculin was used as loading control. (B) Same as in (A) but the analysis was focused on STAT3 and Src signaling pathways. Actin was used as loading control. (C) and (D) Colony assay of MDA MB 231 cells untreated (UNT) or pre-treated for 24 hours with FS-115 or PF-4708671 at the indicated concentration, then counted and seeded at ultra-low density (1000 cells/100 mm dish) and incubated in complete growth medium in the presence of the inhibitors or left untreated. Two weeks later plates were stained with crystal violet and colonies photographed (C) and counted (D). (E) and (F) Anchorage-independent cell growth of MDA-MB-231 cells included in soft agar in 3% wound fluids (UNT), in the presence of PF-4708671 or FS-115 at the indicate d concentration. After three weeks, colonies were photographed (E) and counted (F). Values reported in (D) and (F) represent the mean (± S.D.) of two independent experiments performed in duplicate. * p<0.05; ** p<0.01; *** p<0.001.