Figure 7: Minion and Myomaker are sufficient to induce fusion of non-muscle cells.

(a) Western blot of 10T1/2 fibroblasts expressing Luciferase (Ctrl), Myomaker or Minion. n=2. (b) Retroviral vectors encoding Luciferase, Myomaker or Minion were transduced as indicated into 10T1/2 fibroblasts. All vectors contain IRES-GFP downstream of the gene of interest, causing infected cells to uniformly express GFP. Split-channel grayscale images for GFP and DNA are included. n=3 (eight technical replicates each). (c) Quantification of GFP⁺ syncytia in fibroblasts expressing combinations of proteins as indicated. Fusion index was calculated as the percentage of nuclei found within GFP-positive syncytia containing ≥3 nuclei. Syncytia were scored 24 h after seeding. Each value represents mean±s.d. n=3 (two 1.4 mm × 1.4 mm fields each). Double asterisks: P<0.001, unpaired two-tailed Student’s t-test. (d) Fluorescence images from cell-mixing experiments using differentially labelled 10T1/2 fibroblasts. Cells were serially infected with retroviruses encoding the indicated combinations of Minion, Myomaker, or controls (label omitted for simplicity). CellTrace Violet (cyan) and CellTracker DeepRed (red) dyes were used for labelling. Yellow arrows indicate syncytia containing both DeepRed⁺ cells and Violet⁺ nuclei. White arrows indicate syncytia derived from DeepRed⁺ cells only. n=5 (six technical replicates each). See Supplementary Fig. 19 for split-channel images. (e) Quantification of fusion in d (bottom panels), measured as percentage of DeepRed⁺ syncytia (≥3 nuclei) containing ≥1 Violet⁺ nucleus. Each value represents mean±s.d. n=4 (six 1.4 mm × 1.4 mm fields each). NS, not significant; single asterisk: P<0.05, unpaired two-tailed Student’s t-test. Scale bars, 100 μm (b) and 50 μm (d).