Hpaepic cells

Human alveolar epithelial (HPAEpiC) cells were obtained from ScienCell Research Laboratories (Carlsbad, CA) and cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 (HyClone, Logan, UT) supplemented with 10% fetal bovine serum in a 5% CO₂/95% air incubator. METTL3 knockout HPAEpiC cells were generated via the CRISPR/Cas9 method 37 (link). Briefly, CRISPR/Cas9 technology was used to generate the METTL3^-/- HPAEpiC cell line. First, METTL3 sgRNA (5'-TCAGGAGTTGATTGAGGTAAAGCGAGGTCTAGG-3') was designed by http://crispr.mit.edu and inserted into the pLVX-sgRNA plasmid, and METTL3 sgRNA-containing lentivirus was generated in 293T cells. HPAEpiC cells were seeded into 10 cm tissue culture dishes and cotransfected with METTL3 sgRNA expression lentivirus and Cas9 expression lentivirus for 48 h. Next, HPAEpiC cells were trypsinized using 0.25% trypsin (Gibco) and seeded as single cells into 96-well plates. Cell clones with METTL3 knockout were identified via western blotting and Sanger sequencing.

The U87MG cell line was obtained from the American Type Culture Collection, (Rockville, MD, USA). The cells were cultivated in Eagle’s Minimal Essential Medium (EMEM) enriched with 10% heat inactivated fetal bovine serum (FBS), 100 U/mL of penicillin, and 100 μg/mL of streptomycin at 37 °C in a CO₂ incubator. Human pulmonary alveolar epithelial (HPAEpiC) cells were obtained from ScienCell Research Laboratories (Carlsbad, CA, USA), and cultured according to the manufacturer’s instructions in alveolar epithelial cell medium supplemented with growth factors. NFBTA was dissolved in DMSO and diluted with medium prior to use. The final concentration of DMSO in the cultures was adjusted to <0.1% (v/v). A control experiment was also carried out by replacing NFBTA with the same amount of DMSO (to manage any possible effects of DMSO on the cell growth). Triton X-100 (%1) and Mitomycin-C (0.09 µM) were added to the cells as positive control agents for testing of the cytotoxicity and genotoxicity [60 (link),75 (link)].