Fig 2: SPOP promotes ubiquitin lysine 27-linked poly-ubiquitination of lysine 100 and 127 in Geminin.a 293T cells were transfected with increased Myc-SPOP WT in combination with Flag-Geminin and HA-Ub and harvested for IP and WB. b Control or SPOP knockout 293T cells were transfected with the indicated plasmids and harvested for IP and WB. c 293T cells transfected with plasmids for HA-tagged WT or mutated Ub in combination with other indicated constructs and harvested for IP and WB. d Mass spectrometry analysis reveals Geminin ubiquitination at lysine residues 27, 50, 100, and 127. e, f 293T cells were transfected with Flag-tagged WT or mutated Geminin in combination with other constructs and harvested for IP and WB. Source data are provided in this paper or Mendeley database (10.17632/8n7xt5rkhc.1). Similar results for (a), (b), (c), (e), and (f) panels were obtained in two independent experiments.

Fig 3: GLP/G9a protein is elevated in SPOP-mutant PCa cells in culture and in patient specimens.a Western blots with indicated antibodies in WCL and co-IP samples from 293T cells transfected with plasmids of empty vector (EV) or indicated SPOP mutants and treated with 20 µM MG132 for 8 h. b Western blots with indicated antibodies in WCL and co-IP samples of anti-Flag antibody from 293T cells transfected with the indicated plasmids and treated with 20 µM MG132 for 8 h. c Western blots of WCL from 293T cells transfected with the indicated plasmids. d Western blots of WCL from 22Rv1 cells stably infected with lentivirus expressing EV, WT or mutant HA-SPOP. e, f Representative IFC images of Myc-SPOP and endogenous GLP staining in 22Rv1 cells transfected with Myc-SPOP F102C (e) and the optical density (OD)/nuclear area (pixel) of GLP staining was quantified using ImageJ (f). Data shown means ± SD (n = 50 cells/group). Scale bar, 10 µm. g, h Representative IHC images of GLP and G9a staining in SPOP-WT and Q165P mutant PDX tumors (g) and the quantitative data of GLP and G9a staining (h). Scale bar in 200X images, 100 µm; Scale bar in 400X images, 50 µm. Data shown means ± SD (n = 3 replicates/group). i, j Representative IHC images of GLP and G9a staining in 84 PCa patient specimens (i) and the quantitative data of GLP and G9a staining (j). Scale bar, 50 µm. Statistical significance was determined by unpaired two-tailed Student’s t test in (f, h). Statistical significance was determined by two-tailed Wilcoxon rank-sum test in (j). Experiments in (a–d) were repeated twice. Source data are provided as a Source Data file.

Fig 4: Cancer-associated SPOP mutants fail to interact with and promote the degradation of HDAC6 in cells(A) A schematic illustration of SPOP with MATH and BTB domain and cancer-associated mutations. (B) Immunoblot (IB) analysis of whole cell lysates (WCL) and immunoprecipitates (IP) derived from HeLa cells transfected with indicated plasmids and treated with 10 µM MG132 before harvesting. (C) IB analysis of WCLs and GST pull-down products derived from 293T cells transfected with indicated constructs. (D) IB analysis of WCL derived from melanoma WM2664 cells stably expressed with SPOP WT and SPOP mutants. (E) IB analysis of WCL derived from 293T cells transfected with indicated constructs. (F) IB of WCL and His pull-down from HCT116 cells transfected with the indicated constructs. Cells were treated with 30 µM MG132 for 6 hours and lysed with denature buffer.

Fig 5: Depletion of SPOP enhances the cellular proliferation and migration, which can be reversed partly by additional depletion of HDAC6 in colon cancer cells(A) Immunoblot (IB) analysis of whole cell lysates (WCL) derived from HCT116 infected with the indicated lentiviral shRNAs against SPOP and HDAC6, and subjected to puromycin selection for 72 hours before harvesting. (B) Colony formation assay using HCT116 cells described in Figure 6A. (C) Quantification of colony number of colony formation assays described in Figure 6B. Data were shown as mean ± SD from three independent experiments. *p < 0.05. (D) In vitro scratch assay at 0 and 48 hours using HCT116 cells described in Figure 6A. (E) Quantification of gap closure of in vitro scratch assay described in Figure 6D. Data were shown as mean ± SD from three independent experiments. *p < 0.05. (F) Representative images of migrated HCT116 cells described in Figure 6A in migration assays. (G) Quantification of migrated cells described in Figure 6F. Data were shown as mean ± SD of three independent experiments. *p < 0.05.