D1152

U2-OS (human osteosarcoma) and MDA-MB-231 (triple-negative human breast adenocarcinoma) cells were grown in DMEM (Dulbecco’s Modified Eagle’s Medium with HEPES modification; Sigma, D1152) supplemented with 10% fetal bovine serum (FCS) (Biowest, S1860). CHO cells were cultured in alpha-MEM, supplemented with 5% FCS and L-glutamine.

The adherent cancer cell lines HeLa, HEK293, A549, and HaCaT were obtained from NCCS, Pune. All cells were cultured in high glucose DMEM media (D1152, Sigma-Aldrich) containing 10% FBS and 5% CO₂. We followed the standard protocol^{50 (link)} for the western blotting of the lysates from the mentioned cell lines. Cadherin-23 (HPA017232, Sigma-Aldrich and PA5-43398, Invitrogen), eGFP (A11122, Invitrogen), and His-tag (11965085001, Roche) antibodies were used at the concentration of 1 μg/ml to detect the proteins.
RNA from different cancer cell lines was extracted using RNA isolation kit (Bio Rad) and treated with DNAse using DNAse 1 kit (AMPD1, Sigma-Aldrich). cDNA synthesis was done using a cDNA synthesis kit (Bio Rad). qRT-PCR was performed with the primers probing Cdh23 using the real-time PCR system (CFX96 Bio Rad).

Rats were bred at the Faculty of Chemical and Pharmaceutical Sciences Animal Breeding Facility (University of Chile). NRVM were isolated from the hearts of neonatal Sprague-Dawley rats as previously described (Kuzmicic et al., 2014 (link)). Neonatal pups, between 2–3 days old, were decapitated, and the hearts were extracted and washed with Hank’s buffer (Sigma, H2387) supplemented with NaHCO₃ and HEPES, at 37°C. Cardiac atria were carefully removed, and the remanent ventricular portion was minced. The tissue was then enzymatically digested with type II collagenase (Gibco, 17101–015) (0.02 g/100 mL Hank’s) and pancreatin (Sigma, P3292) (0.06 g/100 mL Hank’s). The cell suspension was pre-plated in a 250 mL culture flask for 2–3 h in DME- (Sigma, D1152) and M199- (Sigma, M2520) containing medium, supplemented with 10% FBS (Biological Industries, 04-121-1A), to obtain a cardiomyocyte-enriched fraction. Cells in suspension were collected and centrifuged at 1,000 r.p.m. for 5 min and then resuspended in 25 mL of DME:M199 (4:1) medium, supplemented with 5% FBS and 10% NBCS (Gibco, 16010–159), to be finally seeded in gelatin 2% p/v (Winkler, GE0820) -coated plates.
All procedures and experiments in animals were performed according to NIH Guide for the Care and Use of Laboratory Animals and approved by the corresponding Institutional Ethics Committee.

Primary cultures of rat neonatal contractile cardiac myocytes (NCMs) were prepared from heart ventricles of 1- or 2-day-old rats, killed by decapitation, minced in a balanced salt solution containing 20 mmol/L HEPES, 120 mmol/L NaCl, 1 mmol/L NaH₂PO₄, 5.5 mmol/L glucose, 5.4 mmol/L KCl, and 0.8 mmol/L MgSO₄ [pH 7.4] and then digested by using pancreatin and collagenase type II as previously described^{6 (link)}. NCMs were purified by centrifugation of the cells at 2800 rpm for 30 min on Percoll (P4937, Sigma-Aldrich) gradients (58.5% and 40.5% in the balanced salt solution). NCMs were seeded at a density 6.5 × 10⁵ cells/well in 6-well plates coated with 10% of collagen (C8919, Sigma-Aldrich) and cultured in a medium containing 4 parts of Dulbecco’s modified Eagle’s medium (DMEM, D1152, Sigma-Aldrich) and 1 part of Medium199 (M199, M2520, Sigma-Aldrich), 10% fetal bovine serum (FBS, 30-2020, ATCC) and 1% penicillin and streptomycin (P/S, 10,000 U/mL) (15140-122, Invitrogen™, Life Technologies) at 37 °C under 5% CO₂ atmosphere.