Fig 2: Gene expression of m6A-methyl-modifying enzymes. MDA-MB-231 and MDA-MB468 cells were cultured for 24 h and 48 h with NO (DETA/NO, 500 μM) and for 10 days (DETA/NO, 100 μM). The expression levels of FTO, ALKBH5, and METTL3 were measured in both cell lines by a, (RT-qPCR), dark blue “control” are the basal expression level of each gene, and b, Immunoblotting (numbers below indicate normalized protein expression level changes). n = 3 separate experiments for a and b. RT-qPCR data are represented as mean ± s.e.m., P-values were determined by unpaired two-tailed Student's t-tests.

Fig 3: NO and FTO regulate the same m6A-mRNA in vitro and in vivo. a, mRNA expression (RT-qPCR) of select m6A-methylated transcripts in untreated MDA-MB-231 cells (light blue bars), NO-treated MDA-MB-231 cells (DETA/NO; 500 μM, dark blue bars), and MDA-MB-231 FTO knockdown cells (purple bars) at 72 h. Methylated genes that are upregulated by NO are in the left panel and those that are downregulated by NO are in the right panel, n = 3 separate experiments. b, Relative m6A-mRNA levels in MDA-MB-231 xenograft tumors from mice that had “NO-producing” tumors (untreated; dark blue bars), and from mice with tumors that did not synthesize NO, (treated with a NOS2 inhibitor aminoguanidine (AG) for 37 days, light bars), ELISA. c, mRNA expression (RT-qPCR) of genes from the same MDA-MB-231 xenograft tumors ( ± AG) for select genes that were m6A-methylated and NO-regulated. Methylated genes that are upregulated by NO are in the left panel and methylated genes that are downregulated by NO are in the right panel. Data are represented as mean ± s.e.m., P-values were determined by unpaired two-tailed Student's t-tests, median line is shown, n = 6 mice/treatment group.

Fig 4: NO inhibits FTO and increases m6A-mRNA in cancer cells. a, Relative abundance of m6A-mRNA in HCC4006 (lung), MDA-MB-231, -468 (TNBC), OVCAR-4 (ovarian), PC-3 (prostate), and U251 (glioblastoma) cancer cell lines that were treated without (light bars) or with (dark bars) NO (DETA/NO 500 μM, 24 h), ELISA. b, Total m6A-mRNA in 2 TNBC cell lines (MDA-MB-231,-468) after 24 h dose course treatment with NO (DETA/NO), ELISA. c, Total m6A-mRNA measured after 10 days of NO treatment (DETA/NO 100 μM), ELISA. d, Representative immunoblot for NOS2 protein in MDA-MB-231 ± DETA/NO (500 μM, 24 h), 231VC (transfected with empty vector control), and 231NOS2+ (transfected with NOS2 gene) cells. e, Accumulative NO synthesis (nitrite and nitrate) in the media of MDA-MB-231 (control) cells, 231NOS2+ (NOS2) cells, or 231NOS2+ cells treated with l-NAME (1 mM) after 24 h. f, Relative abundance of m6A-mRNA in MDA-MB-231 (control) cells, 231NOS2+ (NOS2) cells, and 231NOS + cells treated with l-NAME (1 mM; 24 h), ELISA. g, Relative abundance of m6A-mRNA in MDA-MB-231, and -468 cells after 24 h treatment with NO (500 μM DETA/NO) or the FTO inhibitor meclofenamic acid (MA, 100 μM), ELISA. DETA/NO and MA (IC50 8 μM) both achieve 100 % FTO inhibition at these concentrations. h, Schematic showing how inhibitors of METTL3 (methyltransferase) and FTO affect m6A-mRNA levels. i, Representative immunoblot and densitometry for FTO in MDA-MB-231 cells (control), MDA-MD-231 cells transfected with FTO siRNA (FTO knockdown) or transfected with non-targeting siRNA (transfection control) and j, m6A-mRNA levels in the same cells treated with or without NO (DETA/NO, 500 μM; 72 h), ELISA. k, m6A-mRNA in MDA-MB-231 cells treated NO (DETA/NO 100 μM), or the METTL3 inhibitor cycloleucine (CL, 1 mM), or NO and CL together (n = 4), ELISA. d,i, cell lysates were prepared and immunoblotted with the indicated antibodies, data were independently replicated at least three times, with similar results. a-c,e-g,j n = 3 independent experiments. Data are represented as mean ± s.e.m., P-values were determined by unpaired two-tailed Student's t-tests, median line is shown.