Fig. 3: PKCλ/ι-mediated EZH2 phosphorylation regulates its protein stability. | Nature Communications

Fig. 3: PKCλ/ι-mediated EZH2 phosphorylation regulates its protein stability.

From: Increased translation driven by non-canonical EZH2 creates a synthetic vulnerability in enzalutamide-resistant prostate cancer

Fig. 3

a In vitro phosphorylation of HA-tagged EZH2 by recombinant PKCλ/ι (n = 3 independent experiments). b Identification of EZH2 phosphorylation sites by PKCλ/ι: HA-EZH2, in vitro phosphorylated with recombinant PKCλ/ι, or HA-EZH2 transfected into sgC and sgPRKCI cells were analyzed by MS (n = 1 sample per condition). c In vitro phosphorylation of HA-EZH2WT or EZH2S375/380AA as in (a) (n = 2 independent experiments). d Alignment of the amino acid sequence of human EZH2 (372-383 aa) with orthologs in other species. e EZH2WT or EZH2S375/380AA LNCaP cells were incubated with CHX (50 μg/ml) at indicated time points, and EZH2 levels were quantified (n = 3 independent experiments). f Immunofluorescent staining of EZH2 in EZH2WT or EZH2S375/380AA LNCaP cells and quantification (EZH2WT: n = 91, EZH2S375/380AA: n = 83 cells examined). Scale bars 10 μm. g, h Immunoblots of HA-tagged immunoprecipitates in HEK293T, transfected for the indicated plasmids (n = 2 independent experiments). i Immunoblots in nuclear lysates from sgPRKCI and sgC LNCaP cells (n = 2 independent experiments). j Immunofluorescence staining of pEZH2(S380) in sgPRKCI and sgC LNCaP cells (sgC: n = 459, sgPRKCI: n = 270 cells examined), and quantification. Scale bars 10 μm. k Immunoblots in PtenΔ/Δ, PtenΔ/ΔPrkciΔ/Δ, PtenΔ/ΔRb1Δ/Δ, and PtenΔ/ΔRb1Δ/ΔPrkciΔ/Δ prostate organoids (n = 3 independent experiments). l Immunofluorescence staining for pEZH2(S380), EZH2, PKCλ/ι, SYP and DAPI in prostate tumors from the NEPC model Ptenf/fRb1f/fMYCN+PbCre+ (n = 3 mice per group). Scale bars 200 μm and 20 μm. m Immunofluorescence staining for pEZH2(S380), EZH2, PKCλ/ι, and DAPI in human NEPC PDOs WCM154 (n = 1). Scale bars, 100 μm and 20 μm. n Immunoblots in human adenocarcinoma (MSKPCa3) and NEPC (WCM1262, WCM1078, WCM154) organoids (n = 3 independent experiments). o Immunoblots in control and PRKCI-overexpressed (OE) NEPC PDOs WCM1078 and WCM154 (n = 2 independent experiments). Immunoblot experiments were performed at least two times independently, with similar results. Data shown as mean ± SEM of the biological replicates (e). Two-way ANOVA (e). Two-tailed unpaired Student’s t-test (f, j). Source data are provided as a Source Data file.

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