Fig. 2
Patient-associated mutant forms of SPOP dictate PDK1 stability and oncogenic function. A A schematic illustration of SPOP domains and frequently occurred prostate cancer-associated mutations. B IB analysis of WCL and GST pulldown products derived from HEK293T cells transfected with Flag-PDK1 and GST-SPOP (WT, Y87C, F102C, F133V). Cells were treated with MG132 (10 μM) for 10 h before harvested. C IB analysis of WCL derived from HEK293T cells transfected with indicated constructs. D IB analysis of WCL derived from C4-2 cells stably expressing SPOP WT or mutants. E IB analysis of WCL and His pulldown products derived from HEK293T cells transfected with indicated constructs. Cells were treated with MG132 (10 μM) for 12 h before harvesting. F In vitro ubiquitination assays were performed with commercial E1, E2 and Ub proteins, bacterially purified His-PDK1, as well as SPOP purified from SPOP-overexpressing HEK293T cells. The reaction products were subjected to IB analysis. G, H IB analysis WCL derived from of HEK293T cells transfected with indicated constructs. Where indicated cells were treated with CHX (100 μg/ml) for the indicated time points before harvested. PDK1 protein abundance in (G) was normalized and quantified (H) (mean ± SD, n = 3) (t test), *P < 0.05. I-J IHC staining was performed in prostate cancer tissues, where indicated, WT SPOP tissues displayed weak PDK1 staining, whereas, mutant SPOP tissues displayed intermediate and strong PDK1 staining (I). Scale, top: 100 μm; bottom: 200 μm. The expression of PDK1 was calculated depending on SPOP genetic status and quantified in (J). Chi-squared (χ2) test, P < 0.001. (K-L) C4-2 cell lines stably expressed prostate cancer associated SPOP mutants were infected with shGFP or shPDK1 lentivirus. Cells were selected with puromycin (1 μg/ml) for 72 h to eliminate uninfected cells and used for IB analyses. Resulting cells were subjected to colony formation assays (L, top panel), quantified and plotted (L, bottom panel) (mean ± SD, n = 3) (t test), *P < 0.05, **P < 0.01