Fig. 6: The m6A machinery regulates both YTHDF2 relocation and expression.
a PLA analysis of YTHDF2 associated with IKZF3 in Jurkat cells treated with or without ActD (500ug/ml, 4 h). Scale bar, 10 μm. b Immunoblotting analysis of YTHDF2 in the cytosol and nucleus of Mettl3F/F or Mettl3CKO CD8 T cells stimulated with anti-CD3/CD28 (5 μg/ml, 24 h). c PLA analysis of YTHDF2 associated with IKZF3 in Jurkat-shCtrl and Jurkat-shMETTL3 cells. Scale bar, 5 μm. d PLA analysis of YTHDF2 associated with IKZF3 in primed Mettl3F/F or Mettl3CKO CD8 T cells (5 μg/ml, 24 h). Scale bar, 10 μm. e PLA analysis of Flag associated with IKZF3 in Jurkat cells introduced with Flag-tagged WT or mutant YTHDF2. Scale bar, 10 μm. f Quantification of Ki-67 MFI among METTL3-knockdown and control Jurkat cells with or without YTHDF2 overexpression (OE) (n = 5 independent samples). g Click-it RNA imaging and analysis of nascent RNA synthesis in METTL3-knockdown and control Jurkat cells with or without YTHDF2 overexpression (OE) (n = 5 independent samples). Scale bar, 10 μm. h ATAC-seq tracks of Ythdf2 loci on naïve or activated human T cells (anti-CD3/CD28, 5 h) (GSE116696). i Ythdf2 mRNA levels detected by qPCR in CD8 T cells stimulated with or without anti-CD3/CD28 (5 μg/ml) and ActD (500 ug/ml) for 24 h (n = 5 independent samples). j Ythdf2 mRNA levels detected by qPCR in naïve (0 h) or activated (6 h) WT (n = 5 independent samples) and Ythdf2−249 (n = 5 independent samples) CD8 T cells. k Naïve Ythdf2−249 (n = 3 independent samples) and WT (n = 3 independent samples) CD8 T cells were treated with ActD (500 μg/ml) and RNAs were collected at different time points after ActD treatment. Ythdf2 mRNA levels were measured using qPCR and represented as mRNA remaining after transcription inhibition (TI). l Immunoblotting analysis of YTHDF2 in the cytosol and nucleus of naïve (0 h) or activated (24 h) WT compared with Ythdf2−249 CD8 T cells. Error bars, mean ± s.e.m. One-way (f, i, g) or two-way ANOVA (j) or non-linear regression (k).
