Figure 1

The axis Dll1-Notch1 regulates the in vitro myogenic potential of murine and human MABs. (a–d) Expression levels of Notch1/NOTCH1, Dll1/DLL1 and Hes1/HES1 (Notch1 activation reporter), and WB analysis of Notch1/NOTCH1, its activated form (Notch1/NICD) and Dll1/DLL1 in murine and human MABs over time during spontaneous differentiation in vitro. Expression levels are reported in arbitrary units (AU) as fold change versus day 0. (e, f) Summary array of methylation propensity along upstream CpG islands (one in the murine locus, two in the human), 5′-UTR and 3′-UTR of Dll1/DLL1 genomic loci in murine and human MABs at day 0, day 3 and day 5 of spontaneous differentiation, as assayed by qPCR-based test on genomic fragments enriched in highly methylated DNA. −, unmethylated control, APC promoter; +, methylated control and propensity reference, NBR2 promoter. (g) Immunofluorescence staining on co-cultures of C2C12 myoblasts with murine MABs transduced with lentiviral vectors carrying a GFP tracer and scramble, or anti-Notch1, or anti-Dll1 interfering shRNAs. White arrows indicate chimeric GFP⁺/MyHC⁺ myotubes and MAB myogenic differentiation. Scramble and scramble +γ-secretase inhibitor (gsi) conditions represent the controls of unperturbed and chemically inhibited signaling, respectively. (h) Immunofluorescence staining on differentiated human MABs after transduction with lentiviral vectors carrying a RFP tracer and scramble, or anti-NOTCH1, or anti-DLL1 interfering shRNAs. Presence of MyHC⁺ mono/bi-nucleated myocytes indicate MAB myogenic differentiation. (i, j) Immunofluorescence staining to evaluate the dose-dependent effects of transient Dll1/DLL1 overexpression on the in vitro myogenic differentiation of murine and human MABs, both transduced with lentiviral vectors carrying conditional expression of the ligand under doxycycline (doxy) control; −, 0 μg/ml, basal control; +, 0.1 μg/ml; ++, 1 μg/ml; +++, 10 μg/ml. To confirm Notch signaling involvement in the observed effect, gsi-supplemented control conditions are also shown. To assess the myogenic contribution of murine MABs in co-culture with C2C12, GFP⁺ murine MABs have been used in this experiment. Data in charts are depicted as mean±standard deviation of ≥3 independent experiments. Scale bars indicate 100 μm