Journal: Nature Communications

Article Title: EZH2-induced lysine K362 methylation enhances TMPRSS2-ERG oncogenic activity in prostate cancer

doi: 10.1038/s41467-021-24380-6

Figure Lengend Snippet: a Sequence alignment of ERG domain containing the EZH2 recognized R-K-S motif from diverse species. b Detection of methylated ERG in VCaP cells by immunoblotting with anti-mERG antibody and competition with methylated (M) and non-methylated (C) peptides ( n = 2). c Detection of mERG in VCaP cells by immunofluorescence microscopy with anti-mERG antibody pre-incubated with the specific competitor and control peptides ( n = 2). Scale bar = 20 µm. d Detection of ERG, EZH2, and mERG in VCaP cells by immunofluorescence microscopy ( n = 2). Scale bar = 20 µm. e Detection of ERG and mERG by IB in control and HA-tagged K362A ERG transfected VCaP cells ( n = 2). f In vitro methylation assay with recombinant ERG and EZH2 followed by immunoblots with indicated antibodies (left) and in the presence of the EZH2 inhibitor GSK343 (right) ( n = 2). g Detection of mERG, ERG, and EZH2 by IB in VCaP cells upon EZH2 knockdown by two siRNA (siEZH2 and siEZH2 3′UTR) ( n = 2). h Detection of mERG, ERG, and EZH2 by IB in VCaP cells upon treatment with 10 µM DZNep (H) at indicated time points ( n = 2). i Immunoblots of mERG, ERG, and EZH2 in RWPE1 cells transiently transfected with the indicated ERG and EZH2 expression vectors ( n = 2). j Binding of recombinant ERG and EZH2 determined by microscale thermophoresis (MST). Insert, MST tracing. k Co-IP of ERG and EZH2 in PC3 cells transiently transfected with Ha-tagged ERG expression vector ( n = 2). l Co-IP of ERG and EZH2 in VCaP cells with ERG and EZH2 specific antibodies and control IgG ( n = 2). m Diagram of truncated ERG constructs. n Co-IP and His-pulldown in PC3 cells transiently transfected with the His-ΔN-ERG constructs and immunoblotting with anti-His and anti-EZH2 antibodies ( n = 2). o Diagram of truncated EZH2 constructs. p Binding of Myc-EZH2-∆SET to Ha-ERG assessed by co-immunoprecipitation in PC3 cells transiently transfected with the truncated EZH2 constructs along with ERG plasmid ( n = 2). Molecular weights are indicated in kilodaltons (kDa). Source data are provided as a Source Data File.

Article Snippet: The methyltransferase reaction was first carried out in a 115-μL reaction mixture containing 500 ng of ERG recombinant protein (Origene, TP308093), 500 ng of recombinant PRC2 complex (Active Motif), 100 nM GSK343, and 100 μL SAM Master Mixture (Assay Buffer, MT Enzyme Mixture, MT Colorimetric Mixture and MT SAM).

Techniques: Sequencing, Methylation, Western Blot, Immunofluorescence, Microscopy, Incubation, Control, Transfection, In Vitro, Recombinant, Knockdown, Expressing, Binding Assay, Microscale Thermophoresis, Co-Immunoprecipitation Assay, Plasmid Preparation, Construct, Immunoprecipitation