Figure 5: Minion is specifically required for fusion of skeletal muscle progenitors.

(a) Immunofluorescence of primary embryonic myoblasts isolated from E18.5 Minion^+/+, Minion^Δ/+ and Minion^Δ/Δ embryos, following 3 days in DM. Desmin (green) and DAPI (red). White arrowheads: myotubes; white arrows: elongating myoblasts. n=3 (five technical replicates each). (b) Fusion index of myoblasts in a, calculated as % nuclei in Desmin⁺ myotubes (≥3 nuclei) of total nuclei in Desmin⁺ cells. Asterisk: P<0.05, unpaired two-tailed Student’s t-test. Each value represents mean±s.d. n=4 (two 0.7 mm × 0.7 mm fields each). (c) Western blots of C2C12 myoblasts cultured in GM or DM. Cells were lentivirally infected with either control luciferase targeting shRNA (Ctrl) or serially with two shRNA targeting the Minion 3′UTR (Minion^KD) and cultured in GM or DM for the indicated number of days. n=3. (d) Immunofluorescence images of Ctrl and Minion^KD myofibres following 5 days in DM. MHC (green) and DAPI (red). n=5 (eight technical replicates each). (e) Differentiation index for d, calculated as % nuclei in MHC⁺ cells of total nuclei. NS, not significant, unpaired two-tailed Student’s t-test. (f) Fusion index for d, calculated as % nuclei in MHC⁺ myotubes (≥3 nuclei) of total nuclei. Double asterisks: P<0.001, unpaired two-tailed Student’s t-test. (g) Quantification of myotubes by nuclei number for d. For e–g, each value represents mean±s.d. n=4 (two 0.7 mm × 0.7 mm fields each). (h) Immunofluorescence images of Minion^KD cells expressing either control protein (NanoLuc) or human MINION orthologue, after 5 days in DM. MHC (green) and DAPI (red). n=3 (six technical replicates each). Scale bars, 100 μm.