Fig. 3: Acute depletion of METTL9-DEGRON affects the gene expression profile of mNSCs.

a Schematic depicting METTL9-DEGRON system and relative mechanism of degradation upon dTAG^V-1 supply. b Mettl9 genomic locus and its genetic targeting via CRISPR/Cas9 for Mettl9^Deg generation. On the right, the resulting C-terminally tagged METTL9-DEG protein is shown (aa: amino acid; kDa: kDalton). c Validation of METTL-DEGRON protein expression in Mettl9^Degron mESCs by WB (anti-FLAG and anti-ACTIN antibodies). Black, green and dark green asterisks (*) are the lowest, intermediate and top METTL9 bands, respectively. N = 3 WB (and differentiation) experiments. d Time course expression analysis of METTL9-DEG by WB, after supplying dTAG^V-1 (or DMSO, Ctrl) to mESCs for 1, 3, 5.5, 9 or 24 hours. Similar timepoints and drug concentrations used in N > 3 experiments. e Schematic of experimental strategy for acute METTL9-DEG depletion (by dTAG^V-1) in Mettl9^Deg mESCs and molecular analysis at DIV5. f Top 10 Molecular function GO terms down-regulated in Mettl9^Deg RNA-seq (DIV5). Hypergeometric test: colour scale shows adjusted p values (Benjamini-Hochberg (BH) correction). g Normalised TPM expression of neural marker genes, from Mettl9^Deg RNA-seq (DIV5). Error bars represent mean ± SD of N = 5. h Top down-regulated Cellular Component GO terms in Mettl9^Deg. Hypergeometric test: colour scale shows adjusted p values (BH correction). i Relative bulk 1MH levels (% of total histidine) in Mettl9^WT and Mettl9^KO (left); DMSO- and dTAG^V-1-treated Mettl9^Deg (right), NSCs (DIV6), quantified by mass spectrometry. P values of the t-test (two-sided) are shown in black (P < 0.05) and grey (P ≥ 0.05), respectively; error bars show mean + SE, N = 3.