Fig 1.
Dll1-Fc, Dll4-Fc, and Jag1-Fc alter gene expression in mouse myogenic cells.
(A) Scheme for analysis of Notch ligands-treated mouse muscle stem cells. (B, C) Relative mRNA expression of Notch effector genes (B) and myogenic genes (C) in mouse myogenic cells cultured with Notch ligand (Control-Fc, Dll1-Fc, Dll4-Fc, and Jag1-Fc: 5 ng/μl). Graphs represent average and 95% confidence interval from 3–4 independent experiments. Welch’s t-test was applied with Bonferroni correction. P-values are <0.05 (*) or <0.01 (**).
Fig 2.
Dll1-Fc, Dll4-Fc, and Jag1-Fc retain Pax7 expression but repress activation markers in mouse myogenic cells ex vivo.
(A) Immunocytochemistry for Pax7, MyoD, and Myogenin of mouse myogenic cells cultured with or without Notch ligands. Hoechst staining reveals nuclei. Scale bar = 100 μm. (B) Quantitative analysis of Pax7, MyoD, and Myogenin in the cultured cells. Data are reported as mean and 95% confidence interval of 200–450 cells per staining condition. Statistical significance was assessed by 2-sample test for equality of proportions with Holm method. P-value are <0.05 (*) or <0.01 (**).
Fig 3.
Dll1/4-treatment is not sufficient for retaining regenerative capacity of mouse donor-cells following transplantation.
(A) Experimental procedure for the engraftment of NOTCH ligands-treated mouse myogenic cells. (B) Immunostaining for dystrophin in TA muscles of Dmdmdx-βgeo mice injected with freshly isolated, control-Fc-treated, Dll1-Fc-treated, or Dll4-Fc-treated mouse myogenic cells 2 weeks after intramuscular engraftment. Scale bar = 100 μm. (C) Quantification of dystrophin+ fibers in TA muscle engrafted with freshly isolated (n = 4), control ligand-treated (n = 5), Dll1 ligand-treated (n = 9), and Dll4 ligand-treated cells (n = 7). All data were plotted with the mean from all engrafted mice. Welch’s t-test was applied with Bonferroni correction. P-values are <0.01 (**).
Fig 4.
JAG1 and DLL4 significantly suppress MYOD mRNA levels in human myoblasts.
(A) Scheme for analysis of NOTCH ligands-treated human myoblasts. (B) Relative mRNA expression of NOTCH effector genes in human myoblasts cultured with or without indicated NOTCH ligand (DLL1: 5–20 ng/μl, DLL4: 5–20 ng/μl, JAG1: 5–20 ng/μl). Values are means ± S.D. (n = 3–5). (C) Relative mRNA expression of myogenic genes in human myoblasts cultured with or without the indicated NOTCH ligand. Values are means ± S.D. (n = 3–5). Statistical significance was assessed by non-repeated measures analysis of variance (ANOVA) followed by the Bonferroni test (vs control). P-values are <0.05 (*) or <0.01 (**).
Fig 5.
JAG1 and DLL4 significantly suppress MYOD protein level in human myoblasts.
(A) Immunocytochemistry of human myoblasts cultured with or without JAG1, DLL1, or DLL4 ligand for PAX7 (red) and MYOD (green). The upper panels indicated the transmission images. Scale bar = 100 μm. (B) Quantitative analyses of PAX7 and MYOD protein expression in human myoblasts cultured with or without NOTCH ligands (JAG1: 5 ng/μl, DLL1: 10 ng/μl, DLL4: 5 ng/μl). Data are reported as mean and 95% confidence interval of 150–200 cells per staining. Statistical significance was assessed by 2-sample test for equality of proportions with Holm method. P-values are <0.01 (**). Representative data are shown, and similar results were obtained using different human myoblasts.
Fig 6.
The effect of NOTCH ligands on human myoblast engraftment.
(A) Experimental procedure for the engraftment of NOTCH ligands-treated human myoblasts. (B) Immunostaining for human SPECTRIN (red), anti-human LAMIN A/C (green), and DAPI (blue) in TA muscles of NOD-SCID mice engrafted with non- or JAG1 ligand-treated human myoblasts 2 weeks after transplantation. Scale bar = 100 μm. (C) Quantification of SPECTRIN+ fibers in TA engrafted with control- (n = 5, n = 3), JAG1- (n = 5), or DLL4- (n = 3) treated human myoblasts. All data were plotted with the mean from all engrafted mice. Welch’s t-test was applied.