Rat cardiomyocytes (H9C2) were obtained from Shanghai Institute for Biological Sciences, Chinese Academy of Sciences, and cultured in high-glucose DMEM and 10% heat-inactivated fetal bovine serum (FBS) supplemented with 100 U/mL penicillin and 100 g/mL streptomycin, at 37°C in a 95% air/5% CO2 incubator. Cells were transfected with siRNA using the Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). Gene-specific siRNA (5′-GGGUAAGUCGAGAAGUGUUTT-3′) for Nrf-2 and negative control siRNA were purchased from GenePharma (Shanghai, China). The transfection efficiency of RNA knockdown was estimated 48 h posttransfection by Western blotting.
Neonatal rat cardiomyocytes (NRCMs) were isolated from Sprague-Dawley (SD) rats as described previously [24 (link)]. Briefly, rat hearts were cut into pieces, washed with ice-cold hanks balanced salt solution (HBSS) three times, and incubated with 0.125% trypsin-EDTA for 15 minutes at 34°C for a total of five times. Then, the NRCMs were centrifuged via a differential attachment technique then seeded in six-well culture plates at a density of 2 × 105 cells per well. The isolated NRCMs were grown in DMEM containing 15% FBS supplemented with 100 U/mL penicillin and 100 g/mL streptomycin, at 37°C in a 95% air/5% CO2 incubator.
Neonatal rat cardiomyocytes (NRCMs) were isolated from Sprague-Dawley (SD) rats as described previously [24 (link)]. Briefly, rat hearts were cut into pieces, washed with ice-cold hanks balanced salt solution (HBSS) three times, and incubated with 0.125% trypsin-EDTA for 15 minutes at 34°C for a total of five times. Then, the NRCMs were centrifuged via a differential attachment technique then seeded in six-well culture plates at a density of 2 × 105 cells per well. The isolated NRCMs were grown in DMEM containing 15% FBS supplemented with 100 U/mL penicillin and 100 g/mL streptomycin, at 37°C in a 95% air/5% CO2 incubator.