Figure 5.

Differentiation Is Aborted in Myoblasts Lacking Kinetochore Binding of Mps1

Top: experimental regime showing myoblast treatment and phenotypic assays. Satellite cell-derived myoblasts from Mps1Δ f/f adult mice treated with either Adeno-GFP (control) or Adeno-GFP-CRE (Mps1Δ f/f) virus and subjected to cell-fate assays in either high- or low-serum culture conditions after recovery.

(A) FACS plots showing Annexin-V and 7AAD staining to quantify necrotic and apoptotic cells in control and Mps1Δ f/f myoblasts 24 hr after virus treatment. Experiments were performed in triplicate; n = 3 mice.

(B and C) Representative images (B) and quantification (C) of senescence-associated β-galactosidase (SA-B gal) assays. Arrows highlight senescent cells. Experiments were counted in triplicate; n = 3 mice.

(D and E) Representative images (D) and quantification (E) of low-density cultures from control and Mps1Δ f/f myoblasts switched to low-serum conditions. Cultures were stained with anti-GFP (green), anti-myogenin (white), anti-PAX7 (violet), and DAPI (blue). Experiments were counted in triplicate; n = 3 mice.

(F) Relative transcript levels of myogenic-fate genes in high-serum (proliferation) and low-serum (differentiation) conditions. Triplicate data from n = 3 mice.

(G and H) Images (G) and quantification (H) of control and Mps1Δ f/f myoblasts after switching to low-serum high-density conditions to test myogenic differentiation (anti-myosin heavy chain; green) and fusion potential. n = 3 trials for each experiment from three mice.

(I and J) Immunofluorescence of pRB under differentiation conditions. n = 3 trials for each experiment from three mice.

The scale bars represent 10 μm (B and I), 50 μm (D), and 20 μm (G). Data are presented as mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01, Student’s t test. See also Figure S6.