Fig. 2: Constitutive Mettl9 depletion impairs mESCs neural priming and differentiation.

a Neural differential protocol adopted in this study. Shading indicates the acquisition of mESCs/NSC/NPC identity. b Mettl9 mRNA expression normalised on β-actin (by qPCR), at DIV0, DIV5, DIV10. Error bars are mean ± SD; N = 3. c METTL9 protein expression shown by Western Blot (WB) from Mettl9^WT mESCs DIV0 to DIV10 (anti-METTL9 antibody; kDa: kDalton; N = 1). d Mouse Mettl9 locus and strategy to generate Mettl9^KO mESCs via CRISPR/Cas9 sgRNAs targeting Exon1 (red). e WB from NSC extracts (DIV7) showing endogenous METTL9 expression with an anti-METTL9 antibody (WT; KO: #88 and #90). Blot representative of N = 3, from 3 differentiation experiments. f Representative immunofluorescence (IF) images of Mettl9^WT and Mettl9^KO NSCs (DIV6) with an anti-NESTIN antibody (green) and Hoechst (grey). Scale bar is 10 μm; relative quantification of NESTIN+ cells on the right (t-test, two-sided); error bars are mean ± SD. Quantified cell numbers (n) are shown above each panel (10 fields of view per condition; N = 2 differentiation experiments). g Timepoints of Mettl9^KO mESC neural differentiation (DIV5 and DIV10) analysed by RNA-seq. h Top (10) GO Molecular function terms down-regulated in Mettl9^KO RNA-seq (DIV5). In red, neural-related terms. Hypergeometric test: colour scale shows adjusted p values (Benjamini-Hochberg (BH) correction). i Normalised transcripts per million (TPM) expression of selected basal telencephalic markers. Error bars represent mean ± SE of N = 4. j Top (10) GO Cellular Component terms down-regulated in Mettl9^KO RNA-seq (DIV5; see Methods). Hypergeometric test: colour scale shows adjusted p values (BH correction). k Cell type composition in control WT (E14 or clonal) and Mettl9^KO (#88 and #90) lines inferred by SCADEN deconvolution analysis of scRNA-seq data (DIV10). Asterisk (*) is p = 0.029 (Wilcoxon test; N = 4).