Figure 5. Skp2 ligase inhibitors induce cell cycle changes.

(A) p27-dependent G1 arrest. 501 Mel cells expressing empty vector (shEmpty) or two different shRNAs (shp27-1 or shp27-5) against p27 were (Left) immunoblotted for p27, Skp2, Skp1, and alpha-tubulin or (Center) stained with Propidium Iodide for FACS analysis. Graph represents mean ± STDEV from n=3. *, p < 0.01; **, p < 0.001 compared with shEmpty (one-way ANOVA followed by Dunnett test). (Right) shp27-1 501 Mel cells treated with Vehicle (0.1% DMSO, Veh) or 10 μM inhibitor (C1, C16, C2, or C20), and stained with Propidium Iodide for FACS analysis. Graph represents mean ± STDEV from n=4.

(B) G1 induction by inhibitors. shEmpty 501 Mel cells treated with Vehicle (0.1% DMSO, Veh) or 10 μM inhibitor (C1, C16, C2, or C20) were (Left) immunoblotted for p27, Skp2, Skp1, and alpha-tubulin or (Right) stained with Propidium Iodide for FACS analysis. Graph represents mean ± STDEV from n=4. *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with shEmpty Vehicle (one-way ANOVA followed by Dunnett test).

(C) G1 or G2/M induction by C1 in breast cancer cells. (Left) FACS analysis of T47D and MCF-7 treated with Vehicle (0.1% DMSO, Veh) or 5 μM C1. Graph represents mean ± STDEV from n=4 for T47D and n=3 for MCF-7. *, p < 0.0001 compared with corresponding cell cycle phase in Vehicle (unpaired two-tailed Student’s t test). (Right) FACS analysis of MCF-7 treated with increasing C1 dose. Graph represents mean ± STDEV from n=3. *, p < 0.01; **, p < 0.001 compared with Vehicle (one-way ANOVA followed by Dunnett test).

(D) G1 induction by C2 in prostate cancer cells. FACS analysis of LNCaP cells treated with Vehicle (0.1% DMSO, Veh) or 10 μM C2. Graph represents mean ± STDEV from n=6. *, p < 0.0001 compared with Vehicle (unpaired two-tailed Student’s t test).

(E) p27 protein induction in cancer cells. Immunoblotting for p27, Skp2, Skp1, and alpha-tubulin in MCF-7, T47D, or LNCaP cells treated with Vehicle (0.1% DMSO, Veh), 5 μM C1, or 10 μM C2.

See also Figure S4.