Fig. 1

Increase in the nuclear 6mA levels through nuclear activation of METTL4 expression under hypoxia. a An increase in nuclear 6mA levels was observed in BFTC909 and FADU cells under hypoxia (see “Methods”). The collected results were summarized as the ratios of 6mA/dA in the bar graph (lower panel). Corresponding 6mA dot blots with methyl blue loading controls are shown together with bar graphs. N, normoxia; H, hypoxia. Normoxic condition was used as a control. b Immunofluorescence staining shows the increased nuclear staining of 6mA in FADU cells under hypoxia. Staining of cell nuclei by DAPI and mitochondria by MitoTracker was used as controls. Bar graph indicated the percentage of cells containing nuclear 6mA signals. c Detected 6mA and dA in METTL4-induced gDNAs were verified by product ion conformation spectra (PICS) fit to the spectrum generated from each standard. Parental ion of 6mA was m/z 266 with major daughter ion m/z 150. Parental ion of dA was m/z 252 with major daughter ion m/z 136. d Western blot analysis shows the more prominent nuclear activation of METTL4 levels through nuclear fractionation in two different cell lines. Histone H3 and GAPDH was used as a nuclear and cytoplasmic control, respectively. e Immunofluorescence staining shows the increased nuclear METTL4 expression in cells under hypoxia in BFTC909 and FADU cells. Mitotracker: mitochondria DNA. Cell nuclei were stained by DAPI. f Knockdown of METTL4 abolished the increase in nuclear 6mA levels induced by hypoxia in BFTC909 and FADU cells. Corresponding 6mA dot blots are shown. g In vitro DNA methylation assays show an increase in the 6mA levels by incubating METTL4 with genomic DNAs from BFTC909 or FADU cells. The METTL4 mutant and incubation without SAM were used as controls. Corresponding 6mA dot blots are shown. N, normoxia; H, hypoxia. Normoxic condition was used as a control. The asterisk (*) indicated statistical significance (P<0.05) between experimental and control groups