Fig. 1
From: Cell surface flip-flop of phosphatidylserine is critical for PIEZO1-mediated myotube formation
Defective myotube formation by CDC50A-deficient and ATP11A-deficient myoblasts. a–d Aberrant morphologies of PS flippase-deficient myotubes. a Syncytia formed by WT and CDC50A-deficient or ATP11A-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, CDC50A or ATP11A siRNA were visualized by immunofluorescent staining with anti-MyHC antibody and DAPI. 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 Rescue of morphologies in PS flippase-deficient myotubes by overexpression of PS flippase complex components. e Syncytia formed by WT, CDC50A-deficient or ATP11A-deficient C2C12 myoblasts expressing FLAG-tagged CDC50A, ATP11A, ATP11B or ATP11C were visualized by immunofluorescent staining with anti-MyHC antibody and DAPI in Supplementary Figure 3b. Left: cell fusion evaluated as percentages of syncytia containing ≥50 nuclei. Right: polarized elongation evaluated as percentages of syncytia with aspect ratios ≥3. f Localization of FLAG-tagged proteins expressed in CDC50A-deficient or ATP11A-deficient C2C12 myoblasts. g, h PS exposure on PS flippase-deficient myoblasts. g Flow cytometry analysis of inward translocation (flip) activity of fluorescence-labelled PS (NBD-PS) at the plasma membrane of WT, CDC50A-deficient and ATP11A-deficient C2C12 myoblasts. h Flow cytometry histogram of WT, CDC50A- and ATP11A-deficient C2C12 myoblasts labelled with annexin V-GFP. i, j Mislocalization of cortical actomyosin in PS flippase-deficient myotubes. i Localization of F-actin (phalloidin, red) and NMIIA (anti-NMIIA antibody, green) in WT, CDC50A-deficient and ATP11A-deficient C2C12 syncytia. Arrows indicate syncytia with diminished peripheral NMIIA accumulation. j Cortex vs. cytoplasm ratio of F-actin and NMIIA signals in i. ****P < 0.0001 (Student’s t-test). NS not significant, 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 (f, i)
