Extended Data Fig. 7: Features of YTHDF1/3 PAR-CLIP data and mRNA-seq.
From: O-GlcNAcylation determines the translational regulation and phase separation of YTHDF proteins
(a) Immunoblotting of Ythdf1/3 knockdown HEK293T and HeLa rescued by YTHDF1 or YTHDF3. Labelled transfection conditions were used for YTHDF1/3 PAR-CLIP and RNA-seq. The experiment was performed once. (b) FPKM of YTHDFs in HEK293T (b) and HeLa (c). Ctrl, Ythdf1/3 knockdown. WT, Ythdf1/3 knockdown rescued by WT-YTHDF1/3. Mut, Ythdf1/3 knockdown rescued by YTHDF1/3-Mut. Cumulative distribution log2-fold changes of mRNA input between Ythdf1/3 knockdown and Ythdf1/3 knockdown rescued by WT-YTHDF1/3 (d, f) or YTHDF1/3-Mut (e, g) in HEK293T (d, e) and HeLa (f, g) for non-target (grey) and YTHDF1/3 target (red). Distribution with boxplot and p values were calculated using two-sided t-test. Boxplot bounds depict quartile 1, median and quartile 3, with whiskers at 1.5× interquartile range. n = 58813 genes in total. The experiment was performed with 3 independent replicates. (h) Translation efficiency analysis of N-YTHDF1-λ and N-YTHDF3-λ treated with 1 or 9 g/L glucose in HeLa and the F-luc/R-luc was normalized by respective λ group. Error bars, mean ± s.d., n = 3 biologically independent repeats. Significance was calculated using two-sided t-test. (i) Distribution of translation efficiency of mRNA in HeLa cultured with no glucose or high glucose medium. n = 4655 genes and the experiment was performed with 3 independent replicates. (j) Distribution of log2-fold changes of translation efficiency of YTHDF1 target mRNAs in HeLa cultured with no glucose or high glucose medium. n = 1095 and 2268 genes. The experiment was performed with 3 independent replicates. (k) Distribution of log2-fold changes of translation efficiency of mRNAs containing different m6A numbers in HeLa cultured with no glucose or high glucose medium. n = 1694, 600, 1054 and 1306 genes. The experiment was performed with 3 independent replicates. Distribution with boxplot and p values were calculated using two-sided t-test. Boxplot bounds depict quartile 1, median and quartile 3, with whiskers at 1.5× interquartile range and outlier points.
