Fig. 7: YTHDF2 modulates the expression of E3 ubiquitin ligase ASB2.
From: YTHDF2 governs muscle size through a targeted modulation of proteostasis
a Western blot analysis of ASB2 protein expression in Ctrl and Y2-KO quadriceps at baseline (2 months of age). b Quantification of YTHDF2 protein expression using total protein detection by Ponceau stain as a loading control. qPCR analysis of c total Asb2 mRNA and d cytosolic Asb2 mRNA using the quadriceps muscle of Ctrl and Y2-KO mice. qPCR analyses for Asb2 RNA enrichment in the wild-type quadriceps following RNA immunoprecipitation with e m6A and f YTHDF2, normalized to input RNA. g qPCR analysis for Asb2 following Actinomycin D treatment for the indicated times in H9C2 rat myoblasts transfected with an siRNA pool targeting YTHDF2 (si-Y2) or control non-targeting siRNA (si-Ctrl), normalized to respective t = 0 values. Best-fit values for half-life and decay rate were calculated using least squares regression analysis. h Schematic of muscle overload-induced hypertrophy of the plantaris muscle with siRNA intervention, created in Adobe Illustrator. i Plantaris weight for Ctrl (sham and overload) and siRNA-treated Y2-KO mice 14 days following muscle overload surgery, normalized to tibia length. Biological animal replicates: n = 4 (Ctrl) and 4 (Y2-KO) for panel a–f, and i. Biological cell replicates: n = 3 (si-Ctrl at 0, 0.5, 1, and 2 h) or 4 (si-Ctrl at 4 and 6 h), and 4 (si-Y2 at 0, 0.5, 1, 2, 4, and 6 h) for panel g. Data were presented as the mean ± SEM with the individual biological samples shown. Significance was determined by a two-tailed Student’s t-test for comparisons between Ctrl and Y2-KO mice, and between WT IgG and pulldown antibody (m6A/YTHDF2). Wilcoxon rank-sum test was used to compare means for nonparametric ASB2 protein quantification data. Grubbs’ (ESD) tests were run using GraphPad Prism and outliers were removed from analysis when applicable: *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.
