Extended Data Fig. 1: MBD2c trans-localizes into mitochondria via TOM20 and binds to the noncoding region.
From: Mitochondria-localized MBD2c facilitates mtDNA transcription and drug resistance
a, WB analysis of the subcellular localization of endogenous MBD2c in MCF-10A, HBL-100, PLC, Huh7, THLE-3 and HEK-293T cells. Protein levels were assessed in whole-cell lysate (WCL), mitochondrial (Mito.), cytosolic (Cyto.), and nuclear (Nucl.) fractions, verified by Lamin B, Tubulin, and TOM20 protein levels as WB loading controls. b, WB analysis of the subcellular localization of Flag-tagged MBD2c in MDA-MB-468 and MDA-MB-231 cells. c, Schematic of MBD2c protein structure and selected deletion variants. Subcellular localization of Flag-MBD2c and indicated deletion variants in MDA-MB-468 cells was assessed by WB using anti-Flag antibodies in WCL and purified mitochondrial fraction, validated by Tubulin, Lamin B, and TOM20 protein levels. d, WB analysis of subcellular localization of endogenous MBD2c in MDA-MB-468 cells stably expressing NTC or shTOM20. VDAC was used as the loading control. e, HEK-293T cells were co-transfected with HA-TOM20 and Flag-MBD2c-WT or Flag-MBD2c-ΔC plasmids. Co-IP experiment was conducted using IgG or anti-Flag antibodies, followed by WB analysis. f, ChIP experiments were performed in MDA-MB-468 cells stably overexpressing Flag-MBD2c using IgG or anti-Flag antibodies. The noncoding region (NCR) occupancy by MBD2c was determined by qPCR, with atp8 and mt-co1 as the negative controls (n = 3 samples). g, ChIP-seq experiments conducted in MDA-MB-468 cells stably overexpressing Flag-tagged MTS-MBD2c, further infected with shTFAM-expressing lentivirus by using IgG or anti-Flag antibodies. Integrative Genomics Viewer (IGV) representation of ChIP-seq of MBD2c or BS-seq of 5mC displaying distribution encompassing the entire mitochondrial genome. The red line indicates the sequence of 5mC-modified HSP probes used in Fig. 1i. h, MedIP or ChIP&MedIP assays were conducted in MDA-MB-468 cells stably overexpressing Flag-tagged MTS-MBD2c. MedIP was carried out with IgG or anti-5mC antibodies. 5mC occupancy on NCR was assessed by qPCR (n = 3 samples). Immunoblots are representative of three independent experiments (a-e). In immunoprecipitations, approximately 10% of the supernatant was collected as input and equal volume of input and elution were loaded for WB. Error bars denote the mean ± S.E.M. (f,h). Statistical analyses were performed by two-tailed Student’s t-test (f,h).
