Extended Data Fig. 5: Notch2-induced muscle atrophy is mediated through FoxO signalling pathway.

a–d, Pathway analysis using RNA-Seq data sets that were significantly upregulated in N2^f/f muscle but not in N2^–/– muscle, under hindlimb unloading or diabetic conditions, corresponding to Fig. 1o-q. a, Venn diagram showing DEGs obtained from the comparison between the HU and Cont conditions in GAS muscles from N2^f/f (red circle) and N2^–/– mice (blue circle) (genes with fold change >2.0, q < 0.05, and TPM > 500 were analysed). b, Upregulated pathways obtained from KEGG pathway analysis using genes upregulated in unloaded GAS muscle of N2^f/f but not N2^–/– mice. c, Venn diagram showing DEGs obtained from the comparison between the DM and Cont conditions in GAS muscles from N2^f/f mice (red circle) and N2^–/– mice (blue circle) (genes with fold change >2.0, q < 0.05, and TPM > 500 were analysed). d, Upregulated pathways obtained from KEGG pathway analysis using genes upregulated in diabetic GAS muscle of N2^f/f but not N2^–/– mice. e–g, Effect of constitutive activation of Notch2 with Rbpj ablation on the expressions of atrogenes and FoxO target genes in myofibres. e, HSA^rtTA/+;TRE^Cre/+;Rosa26^LSL-N2ICD/+;Rbpj^flox/flox mice were treated with DOX for 1 week to induce constitutive activation of Notch2 and Rbpj deletion in myofibres (N2-mTg;Rbpj^–/–). Rbpj^flox/flox mice were used as control (Rbpj^f/f). f, qPCR analysis for confirming the overexpression of Notch2 and its target gene Hes1, and the deletion of Rbpj in GAS muscles (n = 4). g. Expressions of FoxO target genes and atrogenes in GAS muscles (n = 4). h,i, Assessment of direct interaction between Notch2 and FoxO transcription factors. h, Satellite cells isolated from EDL muscles of WT mice were transfected with 3xFLAG-N2ICD plasmid by electroporation. Cells were induced to undergo myogenic differentiation for 5 days and harvested as IP samples. i, Immunoblotting analysis for Notch2 (NTM), FoxO1, FoxO3a, and Akt using plasmid-transfected (TF) and non-transfected (Cont) samples immunoprecipitated using FLAG antibody. Total cell lysates were used as input controls. j–l, Effect of Notch2 deficiency on FoxO phosphorylation and localization under atrophic conditions. j, Protein accumulation of phospho-Akt (Ser473), total-Akt, phospho-FoxO1/3a (Thr24/Thr32), total-FoxO3a in GAS muscle of N2^f/f and N2^−/− mice under control, HU, and DM conditions. k,l, Protein accumulation of FoxO1 and FoxO3a in nuclear fractions of GAS muscles from N2^f/f and N2^−/− mice under control and DM conditions. Representative blot patterns (k) and quantitative data (l) were shown. Histone H3 served as an internal control. m–r, Effects of Notch2 deficiency on FoxO1-induced muscle atrophy. m, Satellite cells were isolated from N2^f/f or N2^−/− and transfected with plasmid encoding constitutively active form of human FOXO1 (caFOXO1) or empty vector (Cont) by electroporation. Cells were induced to undergo myogenic differentiation for 4 days and then harvested. n, Gene expressions of Notch2, FoxO1, and FoxO1-target genes including Ccng2 and Gadd45 in myotubes (n = 4). o, Representative images of MyHC immunostaining (scale bars, 50 µm). p,q, Quantitative data of the myotube diameter (p) and the fusion index, which is a proportion of the number of nuclei within MyHC⁺ myotubes (containing more than two nuclei) relative to the total number of nuclei (q) (n = 4). r, Gene expressions of atrogenes. All qPCR data were normalised to Tbp (n = 4). Data represent the means ± s.e.m., and points indicate individual mice. *P < 0.05, **P < 0.01, ***P < 0.001, NS (not significant) as determined by: two-way ANOVA followed by Tukey’s multiple comparisons post-hoc test (l,n,p–r); Student’s two-tailed unpaired t-test (g).

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