Lipofectamine 3000

For gene knockdown, duplex RNA interference (RNAi) oligonucleotides targeting specific genes were designed and synthesized by GenePharma (Shanghai, China), with a scrambled RNA oligonucleotide used as a negative control. The scrambled has the same nucleotide composition as the selected siRNA but no significant homology to any known mRNA. For gene overexpression, cDNAs encoding the full open reading frames of human ALKBH5, WRAP53, USP6, and RALBP1 were sourced from OBiO Technology (Shanghai, China) and Umine Biotechnology (Guangzhou, China). Specifically, RALBP1 and USP6 were engineered to include an N-terminal HA-tag. Additionally, full-length, truncated, and m6A site-mutated variants (wild type, A-T, A-G, A-Del) of WRAP53 were engineered by GenScript (Nanjing, China) and cloned into pcDNA3.1 (-) or pmirGLO vectors for overexpression or dual-luciferase studies, respectively, with an empty vector serving as the control. Cells were transfected with siRNAs using Lipofectamine RNAiMAX (Invitrogen) and with plasmids employing Lipofectamine 3000 (Invitrogen), in alignment with the guidelines provided by the manufacturers. The sequences of siRNAs are detailed in Supplementary Table S3. For stable knockout of ALKBH5, the gRNAs were designed and synthesized by RiboBio (Guangzhou, China), and riboEDIT™ CRISPR-Cas9 system (RiboBio) was used for ALKBH5 knocking out accordingly to the instruction manual. For stable overexpression of ALKBH5, its coding sequence was cloned into a lentiviral vector and co-transfected with packaging plasmids pMD2.G and psPAX2 (Addgene) into HEK 293T cells using the calcium phosphate method. After 48 h, the supernatant containing viral particles was collected and filtered through a 0.45 μm filter. HGC27 cells were infected with the resultant lentivirus at a specified multiplicity of infection. Stable overexpressing cell lines were established by selecting with 1 µg/ml puromycin (Thermo Fisher Scientific, USA) for seven days, followed by expansion and maintenance under puromycin to ensure sustained ALKBH5 expression.

Primary cardiomyocytes were isolated from the hearts of WT mice at P1 using a neonatal dissociation kit and isolation kit (Miltenyi Biotec, Teterow, Germany) according to the instructions. The cardiomyocytes were plated in DMEM supplemented with 10% FBS at 37 °C and 5% CO₂ at a concentration of 5 × 10⁵ cells/ml. For the virus infection experiments, cardiomyocytes were infected with 100 MOI of Ad:CMV:Foxk1 (Ad-Foxk1), Ad:CMV:Foxk2 (Ad-Foxk2), or Ad:CMV:Control (Ad-Ctrl) (Vigene Biosciences, Shangdong, China) in DMEM as previously described in ref. ^{19 (link)}. The purity of neonatal cardiomyocytes was assessed using immunofluorescence staining analysis (Supplementary Fig. 8A). For siRNA experiments, cardiomyocytes were transfected with siRNA-Foxk1, siRNA-Foxk2, siRNA-Hif1α, siRNA-Ccnb1, siRNA-Cdk1 or negative control (NC) using Lipofectamine 3000 (Invitrogen, Waltham, USA) transfection reagent as previously described in ref. ^{27 (link)}. After 48 h, cardiomyocytes were harvested for Western blotting, quantitative real-time polymerase chain reaction, Seahorse XF24 analysis, or proliferation assessment. The siRNA sequences used for targeting genes are listed in Supplementary Table 1 in the Supplementary information.

Cells were cultivated in DMEM medium (Cat No.: #11995, Solarbio Life Sciences, Beijing, China) supplemented with 10% fetal bovine serum (FBS; Cat No.: #10099-141, Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin–streptomycin (Cat No.: #P1400, Solarbio Life Sciences, Beijing, China) at 37 °C within an incubator featuring 5% CO₂ and 95% humidity. The interfering plasmids sh⁃CGREF1 and sh⁃EIF3H1, along with the overexpression plasmid CGREF1, were entrusted to General Biology Anhui Co., LTD. HCC cells in the logarithmic growth phase were inoculated in 6-well plates at a seeding density of 2 × 10⁵ cells/well. After 20 h of cultivation, cell transfection was carried out in accordance with the instructions of Lipofectamine 3000 (Cat No.: #L3000150, Thermo Fisher Scientific, Waltham, MA, USA). Cells were gathered for subsequent experiments.

Cells were co-transfected with pGL3 basic luciferase vector (promoter-less control) or pGL3-BCLAF1 with Renilla luciferase plasmid (pRL-TK) using Lipofectamine 3000 (Thermo Fisher Scientific). Some cells were transfected with siRNA for 48 h before promoter construct transfection. After 48 h, the activities of firefly and Renilla luciferase were measured by Dual-Luciferase Reporter Assay System according to the manufacturer’s instruction (Promega, Madison, WI). The firefly luciferase reading in each transfection was adjusted by normalizing it to the Renilla luciferase reading to account for variations in transfection efficiency. The numerical value was then further normalized relative to that of pGL3-Basic. Assays were performed in triplicate and repeated in three independent experiments.