Anti h3k27me2

Cells were lysed in ice-cold lysis buffer (20 mM Tris–HCl pH 8, 150 mM NaCl, 10 mM MgCl₂, 1 mM EDTA, 0.5% Triton X-100 + protease inhibitors) for 15 min at 4 °C. Samples were then sonicated in a Q800 Waterbath sonicator (QSonica) for 15 cycles of 8 s ON, 15 s OFF. Lysates were cleared after 15 min centrifugation at 10,000 g, and protein concentration was determined using Pierce BCA Protein assay kit (Thermo Fisher). Protein extracts were resolved by SDS-PAGE, blotted to PVDF membranes and probed using the following antibodies: anti-EZH2 (Cell Signaling Technologies, Cat nb#5246, 1:1000), anti-H3K27me3 (Diagenode, Cat nb# C15200181-50, 1:5000), anti-H3K27me2 (Abcam, Cat nb# ab24684, 1:1000), anti-H3K27me1 (Active Motif, Cat nb# 61015, 1:1000), anti-H3 (Abcam, Cat nb# ab1791, 1:2000), GAPDH (Millipore, Cat nb# MAB274, 1:10,000), actin (Abcam, Cat nb# ab3280, 1:5000). Signal was detected using IRDye 680RD and 800CW secondary antibodies following the manufacturer’s instructions (LI-COR). Membranes were acquired on an Odyssey Imager (LI-COR) and quantified using Image Studio (LI-COR). Uncropped images of all western blots are provided in the Source Data file.

For each ChIP reaction, 2 × 10⁶ spheroid cells were fixed in formaldehyde for 15 min at 37°C. Crosslinked chromatin was sonicated to obtain 200–500 bp fragments and immunoprecipitated using anti-KDM4C, anti-H3K27me2, or anti-H3K27me3 antibody (Abcam, China). Normal human IgG was used as a negative control.