Fig 1: SPOP-mutant PCa cells are sensitive to DNA demethylation agent alone or in combination with taxane.a, b Cell viability was measured by MTS assay in 22Rv1 (a) and DU145 cells (b) expressing indicated constructs and treated with 5-AzaC (2 μM) for 5 days. Data shown means ± SD (n = 5 replicates/group). c, d Organoids derived from indicated PDX tumors were cultured with matrigel and treated with DMSO or 5-AzaC (2 or 4 μM) for 7 days followed by photography (c) and quantification (d). Scale bars, 50 μm. Data shown means ± SD (n = 6 fields/group). e, f DU145 cells expressing EV or SPOP-F102C were treated with DMSO, DTX (0.05 nM), 5-AzaC (2 μM) or both for 48 h and harvested for Western blots (e) or subjected to MTS assay (f). Data shown means ± SD (n = 5 replicates/group). g, h DU145 cells expressing EV or SPOP-F102C and treated with drugs as in (e) were subjected to colony formation assay for 12 days followed by photographing (g) and quantification (h). Data shown means ± SD (n = 3 replicates/group). i–k DU145 cells expressing EV or SPOP-F102C were injected s.c. into SCID male mice and treated with vehicle, DTX (5 mg/kg), 5-AzaC (2 mg/kg) or the combination of DTX and 5-AzaC. Tumor volume was measured at indicated time points (i). Tumors were harvested at day-28 and photographed (j), and tumor weight was measured (k). Data shown means ± SD (n = 6 replicates/group). l IHC staining for GLP, G9a, 5mC and Ki-67 was performed. Representative images were taken from each group. Experiments were repeated twice. Scale bar, 50 μm. m–p Quantification of IHC data shown in (l). Percentage of the cells with different intensity of staining (weak, intermediate and strong) for GLP (m), G9a (n) and 5mC (o) were determined. Percentage of Ki-67 staining-positive cells was quantified in (p). Data shown means ± SD (n = 3 xenograft tissues/group). Statistical significance was determined by unpaired two-tailed Student’s t test in (a, b, d, f, h, i, k, p). Experiments in (e) were repeated twice. Source data are provided as a Source Data file.
Fig 2: GLP is a ubiquitination and proteasomal degradation target of SPOP.a Diagram showing the four GLP clones identified from Y2H screen using full-length SPOP as bait. Minimal interacting region shared by positive clones (the region between two dashed blue lines) covers two putative SBC motifs (red rectangles). b Western blots of co-IP samples from 22Rv1 cells treated with 20 µM MG132 for 8 h. c Representative images of proximity ligation assay (PLA) in 22Rv1 cells transfected with indicated plasmids. Scale bar, 10 μm. d Schematic representation of SPOP deletion mutants indicating their binding capability with GLP. e Western blots of WCL and co-IP samples from 293T cells transfected with indicated plasmids and treated with 20 μM MG132 for 8 h. f Western blots of WCL from 22Rv1 cells expressing indicated shRNAs. g RT-qPCR analysis of indicated genes in 22Rv1 cells expressing indicated shRNAs. Data shown means ± SD (n = 3 replicates/group). h Western blots of WCL from 293T cells transfected with the indicated plasmids. i, j Representative IFC images of Myc-SPOP and GLP staining in 22Rv1 cells transfected with Myc-SPOP WT (i). Scale bar, 10 μm. GLP staining was quantified using ImageJ optical density (OD)/nuclear area (pixel) (j). Data shown means ± SD (n = 50 cells/group). k Western blots of WCL from 293T cells transfected with indicated plasmids and treated with DMSO, MG132 (20 μM), bortezomib (200 nM) or chloroquine (50 μM) for 8 h. l Western blots of WCL and co-IP samples from 293T cells transfected with the indicated plasmids and treated with 20 µM MG132 for 8 h. m Western blots of the products of in vivo ubiquitination assays from 22Rv1 cells expressing indicated shRNAs and HA-Ub. n–o 22Rv1 cells expressing indicated shRNA were treated with 50 μg/ml cycloheximide (CHX) and harvested at different time points for western blots (n). Quantification of GLP protein from western blots normalized to actin and then to 0-h time point (o). Statistical significance was determined by unpaired two-tailed Student’s t test in (g, j). Experiments in (b, c, e, h, k, l) were repeated twice. Source data are provided as a Source Data file.
Fig 3: GLP/G9a mediate SPOP mutation-induced hypermethylation independent of its HMTase activity.a Pearson correlation of 5mC and GLP/G9a IHC scores in 84 PCa patient specimens. b–d Western blots of WCL from 22Rv1 cells infected with lentivirus expressing indicated constructs and shRNAs (b). Representative IFC images of 5mC and HA-SPOP F133V staining are shown in (c), and ImageJ was used to quantify the optical density (OD)/nuclear area (pixel) of 5mC signals (d). Data shown means ± SD (n = 50 cells/group). Scale bar, 10 μm. e, f 22Rv1 cells infected with lentivirus expressing indicated constructs and shRNAs were harvested for Western blots with indicated antibodies (e) or used for MTS assay to measure cell proliferation (f). Data shown means ± SD (n = 5 replicates/group). g-i 22Rv1 cells infected with lentivirus expressing EV and SPOP F133V and treated with 2 µM UNC0642 for 24 h for western blots (g). Representative IFC images of 5mC and HA-SPOP F133V staining are shown in (h) and 5mC signals in each group were quantified using ImageJ (i). Data shown means ± SD (n = 50 cells/group). Scale bar, 10 μm. Statistical significance was determined by unpaired two-tailed Student’s t test in (a, d, f, i). Experiments in (b, e, g) were repeated twice. Source data are provided as a Source Data file.
Fig 4: SPOP mutation regulates DNA methylation through GLP/G9a interaction with DNMT proteins.a Diagram showing wild-type GLP, enzymatic inactive mutant C1201A and ANK & NHHC domain double deletion mutant. b Western blots with indicated antibodies in WCL and co-IP samples from 22Rv1 cells transfected with indicated plasmids. c–f 22Rv1 cells infected with lentivirus expressing indicated plasmids and/or shRNAs and transfected with indicated constructs were used for western blot (c), IFC (d) and MeDIP-qPCR (f). ImageJ was used to quantify the optical density (OD)/nuclear area (pixel) of 5mC staining in each group. Data shown means ± SD (n = 50 cells/group) (e). Scale bar, 10 μm. Data shown means ± SD (n = 3 replicates/group) (f). g A working model based on the current findings. Left, WT SPOP recognizes and promotes proteasomal degradation of GLP, thereby destabilizing the GLP/G9a complex, inhibiting GLP/G9a-mediated interaction with DNMTs and DNA methylation, and inducing expression of tumor suppressors such as FOXO3 and inhibition of cell proliferation. Right, PCa-associated SPOP mutations fail to bind and degrade GLP, thereby inducing elevation of the GLP/G9a complex and their interaction with DNMTs, increasing DNA methylation and suppression of expression of tumor suppressors and promoting cell proliferation. However, this process can be reversed by DNA demethylation reagents such as 5-AzaC in SPOP-mutant cells. Statistical significance was determined by unpaired two-tailed Student’s t test in (e, f). Experiments in (b, c) were repeated twice. Source data are provided as a Source Data file.
Fig 5: 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.
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