Myocyte growth medium

Human cardiomyocyte (HCM) (PromoCell GmbH, Heidelberg, Germany) were cultured in Myocyte Growth Medium (PromoCell GmbH, Heidelberg, Germany) at 37 °C in a humidified, 5% CO₂/95% air atmosphere. Passage 3–9 HCMs were used in the experiment. Thereafter, HCMs were then exposed to low glucose (5 mmol/L D-glucose) or high glucose (HG; 35 mmol/L D-glucose) plus palmitate acid (PA; 500 µM, Sigma-Aldrich), with or without the additional 48-hour application of AdipoRon (5, 10 nM). The sequences of the siRNAs were as follows: AdipoR1, 5ʹ-GGACAACGACUAUCUGCUACATT-3ʹ; AdipoR2, 5ʹ- CCAACUGGAUGGUACACGA-3ʹ; and nonspecific scrambled siRNA, 5ʹ-CCUACGCCACCAAUUUCGU-3ʹ (Bioneer, Daejeon, Korea). Human cardiomyocytes in six-well plates were transfected with a final concentration of 50 nM AdipoR1 and AdipoR2 siRNAs using Lipofectamine2000 in Opti-MEM(R) I reduced serum medium (Gibco Invitrogen, Carlsbad, CA) for 24 h and then the medium was changed back to growth medium for additional incubation. After transfection, cells were treated with AdipoRon (50 nM) in high glucose plus palmitate acid media to evaluate the effects of siRNAs on myocyte reactions.

The soft agar colony formation assay was performed using the CytoSelect 96-well cell transformation assay kit (Cell Biolabs, San Diego, CA, USA). Human cervical carcinoma cell line HeLa cells (JCRB Cell Bank, NIBIOHN, Osaka, Japan) were used as a positive control since they are known to form colonies in soft agar medium (Kusakawa et al.^{11 (link)}; Ke et al.^{12 (link)}; Seo et al.^{13 (link)}). The cells were maintained in Eagle’s minimum essential medium (Sigma-Aldrich) supplemented with 10% FBS (Sigma-Aldrich) and 0.1 mM non-essential amino acids (Thermo Fisher Scientific). The primary human cardiomyocytes (HCM) (PromoCell, Heidelberg, Germany) were were maintained in Myocyte Growth Medium (PromoCell). HeLa cells (1%, 100 cells; 0.5%, 50 cells; 0.1%, 10 cells; and 0.01%, 1 cell) were spiked into 1.0 × 10⁴ HCM and grown in soft agar for 10 or 20 days. The cells were lysed and absorbance was recorded on a microplate reader (DS Pharma Biomedical, Osaka, Japan) at a wavelength of 485/520 nm filter set. The experiment was performed in triplicate.

Primary human cardiac myocytes were purchased from PromoCell (the identity of the cell line was not verified; the culture was negative for mycoplasma) and in 75-cm²culture flasks in 20 ml of myocyte growth medium (PromoCell). Cells were detached with accutase, washed once with RPMI containing 200 µM EDDHA and resuspended in PRMI containing 200 µM EDDHA. 40000 cells per well were used for bacterial growth assays as described above.