Fig. 4
METTL7B promoted the m6A modification on ACSL3 mRNA and negatively regulated Erastin-induced ferroptosis via ACSL3. (A) RT-qPCR measurement of ferroptosis-related regulators SW780 and TCCSUP cells, including ACSL3, FANCD2, and FADS2. **P < 0.01 versus shnc. ^^P < 0.01 versus nc. (B) Bioinformatics site SRAMP predicted potential m6A methylation sites in the sequence of ACSL3. (C) Detection of m6A levels among potential binding sites of ACSL3 by m6A-IP-RT-qPCR in SW780 and TCCSUP cells. **P < 0.01 versus IgG IP. ^^P < 0.01 versus IgG IP (nc). (D) The mRNA and protein expression levels of ACSL3 in Erastin-treated SW780 cells with METTL7B overexpression. (E) The mRNA and protein expression levels of ACSL3 in Erastin-treated SW780 cells with METTL7B overexpression and/or ACSL3 knockdown. (F) Proliferation of SW780 cells with METTL7B overexpression and/or ACSL3 knockdown was measured by MTT assay. (G) Lipid ROS production in SW780 cells with METTL7B overexpression and/or ACSL3 knockdown was measured by C11-BODIPY 581/591 staining using flow cytometry. H-I) MDA (H) and intracellular iron content (I) in SW780 cells with METTL7B overexpression and/or ACSL3 knockdown were detected using corresponding commercial kits. **P < 0.01 versus Erastin + METTL7B nc. ^^P < 0.01 versus Erastin + METTL7B oe + nc. shnc: negative control short hairpin RNA. sh1/2: short hairpin RNA 1/2 against METTL7B. nc: empty vector plasmid or small interfering RNA negative control. si: small interfering. oe: METTL7B overexpression plasmid. ACSL3, acyl-coA synthetase long chain family member 3. FANCD2, FA complementation group D2. FADS2: fatty acid desaturase 2. METTL: methyltransferase-like. MTT: 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide. MDA: malondialdehyde