Pcmv ha jmjd3 plasmid

pCMV-HA-JMJD3 plasmid was obtained from Addgene (#24167). Lentiviral shRNAs (Extended Data Table 5) were provided by the Vector Core at the University of Michigan or kindly provided by Dr. Arul Chinnaiyan (University of Michigan). Antibodies including monoclonal anti-EZH2 (1:2000, BD Biosciences, 612667), anti-H3K27me3 (1:1000, Millipore, 07-449), H3K9me2 (1:2000, ab1220, Abcam), H3K9me3 (1:500, ab8898, Abcam), H3K4me1 (ab8895, Abcam), H3K4me2 (ab194678, Abcam), H3K4me3 (ab1012, Abcam), anti-Histone H3 (1:2000, Cell Signaling, 9715), anti-HA (1:200, Santa Cruz Biotechnology, sc-805), anti-DNMT1 (1:250, Abcam, ab13537) were used for Western blotting. Anti-human CXCR3 (1C6) blocking antibody was prepared from mouse hybridoma (ATCC, #HB-12330) by Hybridoma Core at the University of Michigan.

PC-3 cells were plated in 96-well plates (3×10³ cells/well for cell viability assay), 6-well plates (2×10⁶ cells/well for PCR, WB, Senescence) or 10 cm petri dishes (12×10⁶ cells/dish for CHIP) until attached. 2 µg PCMV-HA-JMJD3 Plasmid (Addgene plasmid # 24167) or 50 pmol siRNAs for SUV39H1 and HP1α (GenePharma Co) (Table 1) were transiently transfected into cells using Lipofectamine 2000 reagent (Life technologies) according to the manufacturer’s instructions. 48 h after transfection, cells were treated with various concentrations of Triptolide (π-π technologies) for another 24 h, then collected for downstream experiments.