Cenp a

Flow-sorted chromosomes (10⁵) were cytospun (Cytospin3, Shandon) on to poly(l-lysine) slides at 1,200 r.p.m. for 10 min, as previously described^{36 (link)}. Samples were incubated with blocking buffer (5% normal goat serum in 10 mM Hepes, 2 mM MgCl₂, 100 mM KCl and 5 mM EGTA) for 30 min at RT, and incubated overnight at 4 °C with primary antibodies to Cenpa (catalog no. 2048S, Cell Signaling), H3K9me3 (catalog no. 07-523 or 07-442, Millipore), H3K27me3 (catalog no. ab6002, Abcam or catalog no. 07-449, Millipore), Esrrb (catalog no. PP-H6705-00, Perseus Proteomics) and Sox2 (catalog no. ab97959, Abcam). All antibodies were diluted 1:200 in blocking buffer. Chromosomes were incubated with appropriate secondary antibodies (anti-mouse Alexa 488 (catalog no. A11001, Invitrogen), anti-rabbit Alexa 488 (catalog no. A11008, Invitrogen) or anti-mouse Alexa 568 (catalog no. A11031, Invitrogen)) for 1 h at RT. All secondary antibodies were diluted 1:400 in blocking buffer. Stained chromosomes were mounted in DAPI-containing Vectashield (Vector Laboratories). Wide-field epifluorescence microscopy was performed on an Olympus IX70 inverted microscope using a UPlanApo ×100/1.35 oil objective lens and Micro-Manager software.

Immunoblot analysis was performed from isolated cardiac myocytes or mouse hearts using anti‐GATA4 (Santa Cruz, sc‐1237, diluted 1:500), STAT6 (Abcam, ab44718, diluted 1:500), cyclin A2 (Abcam ab38, diluted 1:400), cenpa (Cell Signaling C51A7, diluted 1:1,000), and anti‐GAPDH (Fitzgerald, 10R‐G109a, diluted 1:6,000) or anti‐actin (Sigma, A2066, diluted 1:10,000) antibodies following standard procedures. Densitometry analysis was conducted with Quantity One software (Bio‐Rad).

For immunoblot analysis, whole-cell lysates were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis, transferred onto nitrocellulose membranes (Bio-Rad), and then probed with the following antibodies: DM1A (α-tubulin, 1:5000, BD), CENP-A (1:1000, Cell Signalling #2186S or Abcam #ab13939), ACA (2 μg ml⁻¹, Antibodies-Online GmbH, 15-235-0001), GAPDH (1:2000, Cell Signalling, 14C10). Not cropped immunoblots are shown in Source Data file. The experiment was repeated twice for each cell line.