Its media supplement

Additional four male DMD^mdx rats at the age of 9 months were anesthetized using isoflurane (2%, inhalation) and killed by cervical dislocation. Cardiomyocytes were isolated from the ventricles of their hearts using a Langendorff setup according to the myocyte isolation procedure described in detail in our previous work (Koenig et al., 2011 (link)). In brief, hearts were rapidly excised, and a cannula was inserted into the aorta for retrograde perfusion with Ca‐free solution containing 0.17 mg/mL Liberase TH (Roche) at 37°C for 18 min. The ventricles were then cut into pieces and incubated on a shaker at 37°C, and Ca concentration was increased to 200 μM over 1 h in five steps. Pieces of digested tissue were then triturated to liberate cardiomyocytes. After a centrifugation step, the cells were resuspended in Minimum Essential Medium (MEM) alpha (Gibco), containing ITS media supplement (Sigma) diluted 1:100, 4 mM l‐glutamine, 50 u/ml penicillin, 50 mg/mL streptomycin, and 25 mM blebbistatin (Sigma). Cells were finally plated on Matrigel (Becton Dickinson)‐coated culture dishes.

Mouse chondrocytes were seeded in 24-well tissue culture plates and cultured in Dulbecco’s modified Eagle medium–F-12 (WAKO) supplemented with 10% fetal bovine serum (NICHIREI), 1% ITS Media supplement (containing insulin, transferrin, and selenous acid; Sigma), and 50 μg/ml ascorbate-2-phosphate (WAKO) for the indicated times. The cells were maintained at 37 °C in a 5% CO₂ incubator with medium changes every 48 h. Data were normalized to the average mRNA level at day 0 (set at 1) and are presented as means ± SEM.

Conditionally immortalized human podocytes were used for in vitro experiments [17 (link)]. Podocytes were grown in RPMI with 10% FCS and ITS media supplement (Sigma–Aldrich) in a humidified incubator, 5% CO₂ at 33°C. Cells were differentiated by transferring approximately 60% confluent cells to 2% FBS media and incubated at 37°C for 2 weeks. Cells were then grown in RPMI with 5 or 25 mmol/l glucose together primed with lipopolysaccharide (LPS, 0.5 µg/ml) [18 (link)] and incubated for 18 h at 37°C followed by 4 h of treatment with MCC950 (10 µM) [11 (link)]. Cells were harvested and RNA was extracted by the TRIzol method and cDNA was synthesized for quantitative RT-PCR as described previously [12 (link)]. Results were expressed relative to the untreated normal glucose control, which was arbitrarily assigned a value of 1. In vitro experiments were repeated three times. Human probe and primer sequences used for quantitative RT-PCR are shown in Supplementary Table S2.