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Myocardiac H9c2 cell (Catalog. No. CRL-1446), a clonal line derived from embryonic rat heart, was from American Type Culture Collection (ATCC, Manassas, VA). The cells were routinely cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma Co., St. Louis, MI) with Dglucose at 4.5 g/L, 10% foetal bovine serum (FBS), 10,000 U/L penicillin, and 10 mg/L streptomycin, in an incubator with 5% CO2, at 37 °C and the medium was changed every 2 days. When confluence reached the cells (between passages 3 and 5) they were subcultured by detaching with 0.25% trypsin–EDTA solution (Sigma Co.) and re-seeding into new plates at a ratio of 1:5 in DMEM with 10% steroid-free FBS (Sigma Co.). Cells at ~75% confluence were cultured in serum-free DMEM, treated with the hormone and its receptor blocker (see below), and subjected to hydrogen peroxide (H2O2, 200 μM, 6 h) treatment.Expression of nuclear NF-κB RelA (p65) in H9c2 myocytes was knocked down by RNA interference using a RelA/p65 siRNA Transfection Kit (GeneResearch Co. Australia) that contained SignalSilence® NF-κB p65 siRNA I (2 μM, mouse specific) and Thermo Scientific DharmaFECT® 1 siRNA transfection reagent. As testing in cultured H9c2 cells by the manufacturer, siRNA (100 nM) complexed with the reagent resulted in silencing (reduction) of mRNA (in QuantiGene® branched DNA/RNA assay) at N90% with little toxicity (cell viability N95% in the alamarBlue® assay). In this study siRNA transfection was performed in accordance with the manufacturer's protocol optimised for use with H9c2 cells in culture. In brief, cells were washed with serum-free DMEM, incubated with the transfection reagent (7 μL in 343 μL DMEM) containing RelA/p65 siRNA (100 nM) for 2–4 h, and continued to culture in serum (10%, steroid-free)-enriched DMEM with the siRNA for 48 h before being ready for the hormonal testing. |
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| Myocardiac H9c2 cell (Catalog. No. CRL-1446), a clonal line derived from embryonic rat heart, was from American Type Culture Collection (ATCC, Manassas, VA). The cells were routinely cultured in Dulbecco's modified Eagle's medium (DMEM, Sigma Co., St. Louis, MI) with Dglucose at 4.5 g/L, 10% foetal bovine serum (FBS), 10,000 U/L penicillin, and 10 mg/L streptomycin, in an incubator with 5% CO2, at 37 °C and the medium was changed every 2 days. When confluence reached the cells (between passages 3 and 5) they were subcultured by detaching with 0.25% trypsin–EDTA solution (Sigma Co.) and re-seeding into new plates at a ratio of 1:5 in DMEM with 10% steroid-free FBS (Sigma Co.). Cells at ~75% confluence were cultured in serum-free DMEM, treated with the hormone and its receptor blocker (see below), and subjected to hydrogen peroxide (H2O2, 200 μM, 6 h) treatment.Expression of nuclear NF-κB RelA (p65) in H9c2 myocytes was knocked down by RNA interference using a RelA/p65 siRNA Transfection Kit (GeneResearch Co. Australia) that contained SignalSilence® NF-κB p65 siRNA I (2 μM, mouse specific) and Thermo Scientific DharmaFECT® 1 siRNA transfection reagent. As testing in cultured H9c2 cells by the manufacturer, siRNA (100 nM) complexed with the reagent resulted in silencing (reduction) of mRNA (in QuantiGene® branched DNA/RNA assay) at N90% with little toxicity (cell viability N95% in the alamarBlue® assay). In this study siRNA transfection was performed in accordance with the manufacturer's protocol optimised for use with H9c2 cells in culture. In brief, cells were washed with serum-free DMEM, incubated with the transfection reagent (7 μL in 343 μL DMEM) containing RelA/p65 siRNA (100 nM) for 2–4 h, and continued to culture in serum (10%, steroid-free)-enriched DMEM with the siRNA for 48 h before being ready for the hormonal testing. |
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