Anti histone h3k27ac

KDM6A expression plasmids pCS2-KDM6A wild type and pCS2-KDM6A catalytically dead mutant (H1146A/E1148A) were gifts from K. Ge (plasmid #17438 and #40619, respectively, Addgene). Lentivirus plasmid pLEX_307 was a gift from D. Root (plasmid #41392, Addgene). Doxycycline-inducible lentivirus plasmids pSLIK-Hygro and entry plasmid pEN_TTmcs were gifts from I. Fraser (plasmid #25737 and #25755, respectively, Addgene). Lentivirus-packaging plasmids psPAX2 and pMD2.G were gifts from D. Trono (plasmid #12260 and #12259, respectively; Addgene). Anti-Myc epitope tag (71D10, Cell Signaling Technology), anti-GAPDH (SC25778, Santa Cruz Biotechnology) were used for Western blot. Anti-Histone H3K4me1, anti-Histone H3K27ac (ab4729 and ab8895, respectively; Abcam), anti-Histone H3K4me3 (39159, Active Motif), and anti-Histone H3K27me3 (07-449, Millipore) were used for ChIP. For Western blot, cells were lysed with radioimmunoprecipitation assay buffer [1% NP-40, 1% sodium deoxycholate, 20 mM tris-HCl (pH 7.6), 150 mM NaCl, 1 mM EDTA, 1 mM Na₃VO₄, 20 mM Na pyrophosphate, 20 mM β-glycerophosphate, 1 mM phenylmethylsulfonyl fluoride (PMSF), and leupeptin (1 μg/ml)]. Cell lysates were subjected to SDS–polyacrylamide gel electrophoresis and Western blot analysis.

For chromatin immunoprecipitation (ChIP)-qPCR experiments, cells were seeded in six-well plates (3 × 10⁵ cells/well) and transfected with siRNAs or negative control oligo-nucleic acids. One day post-transfection, the proteins were cross-linked to DNA using 1 % formaldehyde for 15 min. The harvested cells were washed with 1 × PBS and treated with lysis buffer, and the lysates were subjected to ultrasonic vibration to fragment chromatin. Thereafter, 20 μg of cross-linked fragmented chromatin was immunoprecipitated using a commercial EZ-Magna ChIPTM G kit (Millipore, Billerica, MA, USA). Anti-histone H3K27ac (Abcam ab4729, UK) was used, and goat anti-rabbit IgG was used as the negative control. The retrieved DNA was quantified using real-time qPCR analysis. Three target regions of the GLO1 promoter were quantified using the specific primers listed in Supplementary Table S1. The results were calculated as a percentage of the input DNA using the following formula: percentage input = 2 % × 2 (C[T] 2 % input sample − C[T] IP sample).