Fig. 2

Defective myotube formation by PIEZO1-deficient myoblasts. a–d Aberrant morphologies of PIEZO1-deficient myotubes. a Syncytia formed by WT and PIEZO1-deficient C2C12 myoblasts were visualized by immunofluorescent staining with anti-MyHC antibody (differentiated cells, red) and DAPI (nuclei, cyan). b Left: cell fusion evaluated as percentages of syncytia containing ≥50 nuclei in a. Right: polarized elongation evaluated as percentages of syncytia with aspect ratios ≥3 in a. c Syncytia formed by human primary myoblasts transfected with control or PIEZO1 siRNA were visualized by immunofluorescent staining with anti-MyHC antibody (differentiated cells, red) and DAPI (nuclei, cyan). d Left: cell fusion evaluated as percentages of syncytia containing ≥16 nuclei in c. Right: polarized elongation evaluated as percentages of syncytia with aspect ratios ≥3 in c. e, f Mislocalization of cortical actomyosin in PIEZO1-deficient myotubes. e Localization of F-actin (phalloidin, red) and NMIIA (anti-NMIIA antibody, green) at the cell periphery of WT and PIEZO1-deficient C2C12 syncytia. Arrows indicate PIEZO1-deficient syncytia with diminished peripheral accumulation of NMIIA. f Cortex vs. cytoplasm ratio of F-actin and NMIIA signals in e. g, h Normal cell surface expression of PIEZO1 in PS flippase-deficient myoblasts. g Co-localization of GFP-tagged ATP11A (magenta) and FLAG-tagged PIEZO1 (green) in WT C2C12 myoblasts. Merged images and signal intensities are shown in the bottom panels. h Co-localization of GFP-tagged PIEZO1 (anti-GFP antibody, green) and F-actin (phalloidin, magenta) at the cell periphery of WT, CDC50A-deficient and ATP11A-deficient C2C12 myoblasts. Merged images are shown in the bottom panel. ****P < 0.0001 (Student’s t-test). n sample number. Bar graphs represent mean ± S.E.M. Box and whiskers graph-line: median, box: upper and lower quartiles, whiskers: maxima and minima. Scale bars: 100 μm (a, c), 20 μm (e), 10 μm (g, h)