Figure 6

The effect of GUTK on cell cycle re-entry was diminished after FBXW7 knockdown. LNCaP cells following 5-day serum withdrawal and PC-3 cells at the day of contact inhibition were transfected with 50 nM siRNA duplexes targeting scramble or FBXW7 (#1 and #2) for 48 h (LNCaP) and 72 h (PC-3). FBXW7 α and β isoforms and c-MYC protein in quiescent LNCaP (a) and PC-3 (c) cells were analyzed by immunoblotting. GAPDH served as a loading control. The mRNA levels of FBXW7 were detected by RT-qPCR in quiescent LNCaP (b) and PC-3 (d) cells. TBP was used for normalization. After FBXW7 knockdown, c-MYC protein levels in LNCaP (e) and PC-3 (f) cells, which were induced for cell cycle re-entry with or without GUTK at GI₇₅, were analyzed by immunoblotting. GAPDH served as a loading control. The effect of GUTK on DNA content in FBXW7-knockdown LNCaP (g) and PC-3 (h) cells after release from quiescence for 24–72 h was determined by SYBR green assay. The growth inhibition was calculated using the equation descried under SYBR Green assay in Materials and Methods. The data are expressed as the mean±S.D. of triplicate assays, *P<0.05, **P<0.01, ***P<0.001, compared with the corresponding negative control (NC)