Journal: Cell Death & Disease

Article Title: Notch signaling regulates myogenic regenerative capacity of murine and human mesoangioblasts

doi: 10.1038/cddis.2014.401

Figure Lengend Snippet: 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 μ g/ml, basal control; +, 0.1 μ g/ml; ++, 1 μ g/ml; +++, 10 μ g/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 μ m

Article Snippet: Primary antibodies, dilutions; goat anti-GFP (Abcam), 1 : 500; rabbit anti-RFP (Bio-Connect, Huissen, The Netherlands), 1 : 200; mouse anti-MyHC (DSHB, clone MF20), 1 : 3; rabbit anti-laminin (Sigma-Aldrich), 1 : 300; mouse anti-Sgca (Leica Biosystems, Nussloch, Germany), 1 : 100; mouse anti-human DYSTROPHIN (Leica Biosystems, clone Dys3), 1 : 100; rabbit anti-human sarcomeric ACTININ α (Abcam), 1 : 250.

Techniques: In Vitro, Expressing, Activation Assay, Methylation, Immunofluorescence, Staining, Transduction, Over Expression, Co-Culture Assay, Standard Deviation