Mydgf Recombinant Protein Experiments on Cardiomyocytes

For Mydgf recombinant protein experiments, CMs were isolated from hearts of WT mice at P1 using the Neonatal Heart Dissociation Kit in combination with gentleMACS^TM and Octo Dissociator (Miltenyi BioTec, Teterow, Germany) according to the manufacturer's instructions. CMs were plated into DMEM/F12 medium supplemented with 10% FBS at 37 °C and 5% CO₂. Then CMs were treated with Mydgf recombinant protein and harvested 16 hours later, and analyzed by immunofluorescence and RNA sequencing (RNA-Seq). For inhibition of Akt, CMs were treated with Akt inhibitor LY294002 (Sigma, USA). For siRNA experiments, CMs were respectively transfected with siRNA-Akt, siRNA-c-Myc, siRNA-FoxM1 or negative control (NC) using Lipofectamine 3000 (Invitrogen, Waltham, USA) transfection reagent as previously described 22 (link), harvested 48 hours later, and analyzed by western blot, immunofluorescence, or quantitative real-time Polymerase Chain Reaction (qRT-PCR). The siRNA-Akt sequences were: 5′-GCUACUUCCUCCUCAAGAATT-3′, 5′-UUCUUGAGGAGGAAGUAGCTT-3′, the siRNA-c-Myc sequences were: 5′-CCGUACAAGCCCUAUUUCAUTT-3′, 5′-AUGAAAUAGGGCUGUACGGTT-3′, the siRNA-FoxM1 sequences were: 5′-GCAAAUUUCCAGCCGGAAUTT-3′, 5′-AUUCCGGCUGGAAAUUUGCTT-3′, and the NC sequences were: 5′-UUCUCCGAACGUGUCACGUTT-3′, 5′-ACGUGACACGUUCGGAGAATT-3′.