Figure 4. Evaluation the interconnection among AR-V7, PIP5K1α and CDK1 in the nuclear and cytoplasmic compartments of 22Rv1 cells and the mechanisms of the action of ISA2011B on AR-V7.

(a). Cytoplasmic (Cyto) and nuclear (Nuc) fractions were separated from 22Rv1 cells and were subjected to immunoprecipitation (IP) assay. Antibody to PIP5K1α was used to pull down the immunocomplexes, and antibody to IgG was used as a negative control. Antibodies against PIP5K1α, AR-V7, AR and CDK1 were used for immunoblot analysis (IB). The cell lysates from cytoplasmic and nuclear fractions were used as “Input” controls as indicated in the left panel. β-tubulin and lamin B were used as controls for the cytoplasmic vs. nuclear fractions. (b). Evaluation of ISA-2011B-induced degradation of AR-V7 protein at PIP5K1α-dependent fashion using immunoblot analysis. Expression of PIP5K1α, AR-V7, AR and CDK1 was examined in control 22Rv1 cells, and in cells treated with ISA-2011B or MG132 alone, or ISA-2011B and MG132 together. (c, d and e). The effect of ISA-2011B on the subcellular expression of AR-V7, AR, PIP5K1α and CDK1 in 22Rv1 cells. The representative blots from the same membrane is shown. The expression of these proteins in cytoplasmic (cyto) and nuclear (nuc) compartments of 22Rv1 cells treated with vehicle control (−) or ISA-2011B (ISA-2011B+). Total cell lysates were used as controls (Total). The blots of β-tubulin and Lamin B used as controls were from the same detection as shown in b, c and d. (f). CDK1 was depleted by transfecting 22Rv1 cells with CDK1 siRNA or scramble control (Ctrl). The effect of CDK1 knockdown on PIP5K1α and AR-V7 in 22Rv1 cells is analyzed using immunoblot analysis. The representative immunoblots from the same membrane were used. The blots of β-tubulin used as controls were from the same detection as shown in f. (g and h). The side-by-side comparison of the effect of enzalutamide and ISA-2011B on CDK1 and AR-V7. Data is presented as mean of three independent experiments (±SD). ***p < 0.001.