Cell transfection was performed before H/R and/or CA treatment. Pooled siRNAs targeting Nrf2 (#1, #2 and #3) were used to effectively silence Nrf2 and eliminate off-target effects. The siRNA sequences were as follows: Nrf2#1, forward 5'-GGAAGUCUUCAGCAUGUUATT-3' and reverse 5'-UAACAUGCUGAAGACUUCCTT-3'; Nrf2#2, forward 5'-CGAGAAGUGUUUGACUUUATT-3' and reverse 5'-UAAAGUCAAACACUUCUCGTT-3'; Nrf2#3, forward 5'-GGGUUCAGUGACUCGGAAATT-3' and reverse 5'-UUUCCGAGUCACUGAACCCTT-3'; non-targeting negative control (NC), forward 5'-UUCUCCGAACGUGUCACGUTT-3' and reverse 5'-ACGUGACACGUUCGGAGAATT-3' (Beijing Tsingke Biotech Co., Ltd.). H9c2 cells were seeded in 6-well plates at 1×105 cells/well and were treated at 37°C with 5% CO2 until they reached 70-80% confluence.
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