Figure 3. Loss of VCP induces PDAC cell death.

(A) Cells were reverse transfected with non-specific (siNS) control or two different siRNAs targeting VCP (siVCP #1 or siVCP #2) for 72 hours. Immunoblot analyses were performed to determine knockdown efficiency. Representative of three biological replicates that correspond to panels B and C. (B) Percentage of cells in the specified phases of the cell cycle was determined by propidium iodide staining and flow cytometry following 72 hours of reverse transfection using siNS, siVCP #1, or siVCP #2. Data shown are the mean ± SEM of three independent experiments. Two-way ANOVA followed by Dunnett’s multiple comparison test was used to determine statistical significance. ^*p < 0.05. (C) Cells were transiently transfected with siNS, siVCP #1, or siVCP #2 for 72 or 120 hours. Percentage of cells undergoing apoptosis was determined by FACS analysis of cells labeled with Annexin-V and propidium iodide. All treated populations were normalized to their respective siNS control. Data shown are the mean ± SEM of three independent experiments. Two-way ANOVA followed by Dunnett’s multiple comparison test was used to determine statistical significance. ^**p < 0.0001 and ^***p < 0.0005. (D) Percentage of cells in the specified phases of the cell cycle was determined and analyzed as in panel B, following 72 hours of treatment using CB-5083 (0, 125, 250, 500 nM). ^*p < 0.05, ^**p < 0.01 and ^***p < 0.005. (E) Cells were treated with VCPi CB-5083 (125, 250, or 500 nM) for 72 or 120 hours. Percentage of cells undergoing apoptosis was determined and analyzed as in panel C, except that each respective control was 0 nM CB-5083, i.e., DMSO vehicle only. ^*p < 0.05, ^**p < 0.005, ^***p < 0.001, and ^****p < 0.0001.