Phalloidin atto 647n

The experiments were performed using a Leica TCS SP5 STED microscope (Leica Microsystems, Mannheim, Germany), through a 100 × , 1.4 NA PL APO CS oil objective (Leica Microsystems), as described^{48 (link)}; or using a modified Quad Scan STED Microscope (Abberior) with a 100 × , 1.4 NA UPlanSApo oil objective (Olympus). Actin was labeled using ATTO647N phalloidin (Sigma Aldrich), using a previously published protocol^{49 (link)}. ATTO647N was excited using a pulsed diode laser at 635 nm (Leica Microsystems), and depleted using a 750 nm wavelength, provided by a Spectra-Physics Mai Tai tunable laser (pulsed at 80 MHz; Titanium Sapphire, Newport Spectra-Physics, Irvine, CA). For all stainings 3 independent experiments were analyzed and the total number of cells are presented in Supplementary Table S2.

Cells were fixed with 4% PFAH for 10 min at RT and simultaneously permeabilized and blocked with 0.3% Triton in 30% horse serum (Gibco) for 10 min at RT. Following antibodies and antibody dilutions were used: JMJD1a (12835-1-AP, Proteintech, 1:100), YAP/TAZ (sc-101199, Santa Cruz Biotechnology, 1:75), JMJD1a (sc-376608, Santa Cruz Biotechnology, 1:100), Atto-Phalloidin-647N (65906, Sigma, 1:200), Collagen I (NB600-408, Novus, 1:100) and Fibronectin (F3648, Sigma, 1:400).

Direct and indirect immunofluorescence staining were performed on a 6 μm cryosection to evaluate the effect of decellularization on the ECM composition and to confirm the absence of nuclei. Native pericardium was used as the control. Before staining, samples were fixed in 4% (w/v) paraformaldehyde (PFA) supplied by Bioptica.
The primary antibodies used were collagen I (1:100, C2456; Sigma), elastin (1:50, ab21610, Abcam), and collagen IV (1:200, ab6586, Abcam). The controls were incubated with 1% (w/v) bovine serum albumin, instead of primary antibodies. In order to reveal the primary antibody binding, secondary antibodies were applied in separate: goat-antimouse Alexa Fluor 555 (1:300, A21422; Invitrogen) and goat anti-rabbit Alexa Fluor 555 (1:300, A27039; Invitrogen). Nuclei were stained by 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), following the producer’s instructions. Filamentous actin was fluorescently labelled with Phalloidin–Atto 647N (1:200, 65906, Sigma-Aldrich, St. Louis, MO, USA).
Images were acquired with an epifluorescence microscope Leica AF6000, connected to a Leica DC300 digital camera and equipped with LAS AF Software (Leica Micro-System, Wetzlar, Germany). Image processing was performed using the open-source ImageJ software (NIH, Bethesda, MD, USA).

Sterilized coverslips were pre‐coated with 0.01% Poly‐l‐Lysine (P4832, Sigma) for 5 min and then washed with PBS and dried for 2 h. 1 × 10⁵ HCT116 R248W cells were added and incubated for 72 h. Cells were fixed for 15 min with Pierce™ Methanol‐free 16% Formaldehyde (Thermo Fisher) diluted to 4%, washed with PBS, permeabilized 2 min with 0.2% Triton X, washed, and blocked for 60 min. Blocking buffer contained 2% Bovine Serum Albumin (BSA), 5% glycerol, and 0.2% Tween‐20 in PBS. Primary antibody D7O8N against MRP1 diluted 1:200 (14685S, Cell Signaling, USA) was prepared in blocking buffer and incubated at 4°C over night. Coverslips were washed with PBS, anti‐rabbit (A‐110008, Thermo Fisher) and anti‐rat (A‐110006, Thermo Fisher) Alexa Flour^® 488 secondary antibodies were diluted 1:500 in blocking buffer together with Phalloidin‐Atto 647N 1:500 (65906‐10NMOL, Sigma) and incubated for 1 h. Coverslips were washed and mounted with VECTASHIELD HardSet Antifade Mounting Medium with DAPI (H‐1500 Vector Laboratories). Next day samples were imaged by Zeiss AxioObserver Z1‐inverted microscope equipped with Axiocam 506 mono camera using the 63× oil immersion lens and processed using ZEN software by Zeiss.

At the end of each experiment, the hearts were collected and fixed overnight at 4°C in 2% paraformaldehyde. They were then rinsed in PBS and equilibrated in 30% sucrose before embedding in Tissue-Tek OCT compound (Sakura Finetek Europe B.V.) and cryo-sectioned at a thickness of 16 µm. The immunohistochemistry procedures were performed as previously described [23 (link)]. The following primary antibodies were used: rabbit anti-MCM5 at 1 : 5000 (kindly provided by Soojin Ryu, Heidelberg), mouse anti-p-Histone 3 at 1 : 200 (Millipore, Clone 3H10), rabbit anti-Mef2 at 1 : 100 (Santa Cruz Biotech SC-313), rabbit anti-DsRed (Clonetech, 632496) at 1 : 200 and rat anti-BrdU at 1 : 100 (Abcam, ab6326). To visualize the cardiac muscle, sections were incubated for 30 min with Phalloidin-Atto 647N (Sigma-Aldrich) at a dilution of 1 : 500. For the BrdU immunostaining, the slides were incubated in 2 M HCl in PBS with 0.3% Triton-X for 45 min before the immunohistochemistry procedure. The Alexa-Fluor-conjugated secondary antibodies (Jackson Immunoresearch) were used at 1 : 500, and DAPI was used at 1 : 2000. Haematoxylin and eosin (H & E) staining was performed as previously described [15 (link)].