Downregulation of MLF1 safeguards cardiomyocytes against senescence-associated chromatin opening
Previous studies found that MLF1 mRNA levels tended to decline in aging hearts. Functional studies found that knockdown of MLF1 ameliorated the drug-induced cellular senescence phenotype. Here, we apply CUT&Tag to explore the underlying molecular mechanisms. Overall design: Cardiomyocyte AC16 cells were cultured in DMEM/F12 . DMEM/F12 supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 100 U/mL penicillin, and 100 µg/ml streptomycin. Medium without FBS was replaced before transfection. Negative control siRNA and Mlf1 siRNA or SUZ12 siRNA were transfected into AC16 cells at ~70% confluence using jetPRIME® (Polyplus-transfection, NY, USA). After 12 h of transfection, fresh complete medium was replaced. Utilizing the Hyperactive® Universal CUT&Tag Assay Kit for Illumina (TD903), we performed CUT&Tag experiments as follows: AC16 cells were harvested at room temperature and incubated with ConA Beads for 10 minutes. They were then incubated with anti-MLF1 (ABclonal, A13329), anti-SUZ12 antibody (CST, 3737S, 1:50), anti-EP300 (CST, #57625), anti-H3K27ac (CST, #4353S), or anti-H3K27me3 (CST, #9733) antibodies overnight at 4°C. On the next day, the samples were incubated with a secondary antibody at room temperature for 1h, followed by incubation with pA/G-Tnp for 1h.  After DNA purification with the MinElute kit, and the PCR was performed to amplify the library for 12 cycles and libraries were purified with the MinElute kit. Amplified  libraries were sequenced on the Illumina Novaseq 6000 platform.