Skip to main content
. 2021 Jul 16;40(35):5379–5392. doi: 10.1038/s41388-021-01914-2

Fig. 3. ARVib inhibits AR/AR-V7 protein expression via HSP70/STUB1-mediated ubiquitin-proteasome system regulation.

Fig. 3

A C4-2B MDVR cells were treated with 50 µg/mL cycloheximide with or without ARVibs, after 0, 2, 4, and 8 h, whole-cell lysis was collected and subjected to western blot, half-life of AR-V7 was calculated. B C4-2B MDVR cells were treated with 1 μM Nic, ARVib-7 or ARVib-31 for 16 h with 5 μM MG132 for 6 h, total cell lysates were immunoblotted with anti-AR-V7 and AR antibodies. C C4-2B MDVR cells were treated with ARVibs, whole-cell lysis was immunoprecipitated with AR antibody and blotted with ubiquitin and AR antibodies. D C4-2B MDVR cells were treated with different doses of Nic, ARVib-7, or ARVib-31 for 24 h, whole-cell lysates were collected and subjected to western blot. E GSEA demonstrates strong enrichment of the HSP70 inhibition signature in resistant cells treated with ARVibs. The signature was defined by genes with significant expression changes by HSP70 inhibition in prostate cancer cells [27]. F 293 cells were co-transfected with AR-V7, HSP70, and Flag-STUB1 for 3 days, and then treated with 1 μM Nic, ARVib-7, or ARVib-31 for 24 h, AR-V7 and STUB1 were visualized by dual immunofluorescence staining. White arrows indicate the typical staining of cells in each group. Nuclei were stained by DAPI. Scale bar 20 µm. G AR-V7, HSP70, and Flag-STUB1 were overexpressed in 293 cells, which were then treated with DMSO, Nic, ARVib-7, ARVib-31, or VER for 16 h. Total cell lysates were immunoprecipitated with anti-AR-V7 antibody and immunoblotted with anti-HA-Ub, Flag-STUB1, and AR-V7 antibodies. H CWR22Rv1 cells were treated with DMSO, 0.5 or 1.0 μM ARVib-7 for 16 h and 5 μM MG132 for additional 6 h, cytosolic and nuclear proteins were extracted and subjected to western blot. I CWR22Rv1 cells were treated with DMSO, 0.5 or 1.0 μM ARVib-7 for 16 h and 5 μM MG132 for additional 6 h, cytosolic and nuclear protein were extracted and immunoprecipitated with AR-V7 antibody and blotted with anti-Ub and AR-V7 antibodies. J CWR22Rv1 cells were transiently transfected with STUB1 siRNA for 3 days, and then treated with different doses of ARVib-7 for 16 h. Whole-cell lysates were collected and subjected to western blot. K CWR22Rv1 cells were transiently transfected with STUB1 siRNA for 3 days and then treated with different doses of ARVib-7 for 3 days, total cell numbers were determined. *p < 0.05. Results are the mean of three independent experiments (±S.D.).