Overexpression and Knockdown of Key Glioma Genes

The overexpression plasmids of ASS1 and METTL14 were synthesized and cloned into pcDNA3.1 (Invitrogen, China). The short hairpin RNAs (shRNAs) targeting ASS1, METTL14, and YTHDF2 were obtained from GenePharma (Shanghai, China). The shRNAs and corresponding negative controls were synthesized and cloned into the pGLVH1/GFP/Puro vector (GenePharma, China). Lipofectamine 3000 (Invitrogen, USA) was used to transfect the plasmids into glioma cells, according to the manufacturer’s protocol [22 (link)]. For overexpressing of ASS1, 293 T cells (4 × 10⁵/well) were cotransfected with 4 μg pcDNA3.1-ASS1 by Lipofectamine 3000. 48 hours later, lentiviruses were harvested. The glioma cells were infected with LV-ASS1 with 8 mg/mL polybrene by ViraPower Packaging Mix (ThermoFisher). Stable cell lines were obtained by treatment with 5 μg/mL puromycin (Sigma Aldrich) for 7 days.