Figure 3. Inducible YTHDC1 knockout impairs satellite cell (SC) activation/proliferation.

(A) Left: EdU (red) staining of ASC-24 hr and ASC-48 hr from inducible knock out (iKO) and Ctrl mice. Scale bar = 200 μm. Right: quantification of the percentage of EdU+ cells. n = 3 mice per group for ASC-24 hr, n = 4 mice per group for ASC-48 hr. (B) Left: EdU (red) staining of EDL myofibers isolated from Ctrl or iKO mice and cultured for 48 hr. Scale bar = 100 μm. Right: quantification of the percentage of Pax7+ EdU+ SCs. n = 3 mice per group. (C) Top: immunofluorescence (IF) staining of Pax7 (green) and MyoD (red) of freshly isolated SCs (FISC) (0 hr), ASC-24 hr and ASC-48 hr from Ctrl and iKO mice. Scale bar = 100 μm. Bottom: quantification of the percentage of Pax7+ MyoD+ cells. n = 3 mice per group. (D) Top: IF staining of Pax7 (green) and MyoD (red) on EDL myofibers at 0 hr (freshly isolated), 24 hr, and 48 hr. Scale bar = 100 μm. Bottom: quantification of the number of Pax7+ MyoD+ cells per fiber (n = 4 mice per group for 0 and 24 hr, n = 5 mice per group for 48 hr). (E) Schematic illustration of in vivo EdU assay in Ctrl and iKO mice. (F) Top: EdU staining of the above freshly isolated and fixed SCs at 3 dpi. Scale bar = 200 μm. Bottom: quantification of the percentage of EdU+ cells in iKO vs. Ctrl. n = 3 mice per group. (G, H) Left: RNA-seq was performed in ASC-24 hr or -48 hr from iKO and Ctrl mice. Volcano plot showing the down- and upregulated genes in iKO vs. Ctrl. Right: GO analysis for the downregulated genes. (I) Two independent mAID-YTHDC1 cell lines were treated with DMSO or 5-Ph-IAA (IAA) for the indicated time. EdU assay was performed and the percentage of EdU+ cells was quantified at the designated time points. n = 3 replicates. (J) WT-YTHDC1, or a single (W429A), or a triple (K362A, S363A, and N364A) mutant of YTHDC1 lacking m6A binding ability was transfected with the mAID-YTHDC1 cells then treated with IAA. mAID-YTHDC1 cells treated with DMSO or IAA were used as control. EdU assay was then performed and the percentage of EdU+ cells was quantified. n = 3 replicates. Bars represent mean ± SD for all graphs. Statistical significance was determined using a two-tailed Student’s t-test.

Figure 3—figure supplement 1. Analyses of activation/proliferation defect of satellite cells (SCs) and C2C12 myoblasts upon YTHDC1 loss.

(A) ASCs from Ctrl or inducible knock out (iKO) were cultured for 24, 48, and 72 hr and light microscopy images are shown. (B) Quantification of cells per view at the above time points. n = 2 mice per group. (C) Western blotting showing the expression of YTHDC1, Pax7, and MyoD in ASC-48 hr from Ctrl and iKO. Histone H3 was used as a loading control. (D) Schematic illustration of generating a C2C12 cell line with inducible YTHDC1 degradation using the auxin-inducible degron (AID2) system. (E) Western blotting showing the degradation of YTHDC1 after treating mAID-YTHDC1 clone 1 or 2 with 1 μM 5-Ph-IAA (IAA) for 8 hr. DMSO treatment was used as a control. (F) The above YTHDC1 degradation was confirmed by examining the mCherry signal after treating the cell with 1 μM IAA or DMSO for 24 hr. (G) EdU staining of clone1, clone2, and control cells treated with DMSO or IAA for the designated time points to show the reduced cell proliferation upon YTHDC1 degradation. (H) C2C12 expressing Ostir1 (no AID knock-in) was used as control (Ctrl) to show that IAA small molecule does not affect cell proliferation. Quantification of Edu +percentage of C2C12 (without AID knock-in) treated with DMSO or IAA for the designated time. Bars represent mean ± SD for three views. (I) Western blots showing the degradation of endogenous AID-YTHDC1 and overexpression of WT-YTHDC1, m6A binding mutants of YTHDC1 (W429A, triple mutant). GAPDH was used as the loading control.