Fig. 1: METTL3/METTL14 knock-out impairs nucleoli integrity and function in human ESCs.

a Left panel, Representative images for morphology of wild type (WT), METTL3- or METTL14- deleted H1 hESCs from three independent images (n = 3). Scale bar, 50 μm. Right panel, western blot of METTL3 or METTL14 in indicated cells. b Membrane staining of the indicated cells. The cells were stained by DiD for membrane and DAPI for nuclei. Scale bar, 10 μm. c Left panel, Heatmap on the expressions of selected pluripotent genes in the indicated cell lines. Middle panel, Heatmap of down-regulated genes or up-regulated genes in METTL3- or METTL14-deficient hESCs compared with WT H1 hESCs. Right panel, top 10 enriched GO-BP terms in downregulated genes. d Cell cycle analysis of wild type (WT), METTL3-deficient, and METTL14-deficient hESCs. e Upper panel, immunostaining on the nucleolus marker NPM1 in the indicated cell lines. Scale bar, 10 μm, and 3 μm. Lower panel, transmission electron micrographs of nuclear and nucleolar morphology of WT, METTL3-deficient, or METTL14-deficient hESCs. Nucleoli regions were enlarged as indicated. Scale bars were indicated. f The quantification of nucleolus number, nucleolar diameter, and size in the indicated cell lines. The nucleoli were labeled by NPM1 and calculated manually. g qRT-PCR analysis on the expression of pre-rRNA in the indicated cell lines. Wild type H1 hESCs serve as control. h Polysome profiling in wild type (WT) hESCs and DOX dependent exogenous METTL3 expression in METTL3^−/− hESCs (METTL3-OE/KO) upon withdrawal of DOX treatment at day 18. The profiling was analyzed using by sucrose density-gradient ultracentrifugation. i Western blot analysis and quantification on P53 the indicated cell lines. The data in (d, g, and i) represent mean ± SD from three independent experiments (n = 3), and in (f) represent mean ± SD from five independent experiments (n = 5). Statistical analysis, unpaired two-tailed Student’s t-test. **P < 0.01.