c2c12 myoblasts  (ATCC)


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    ATCC c2c12 myoblasts
    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 <t>C2C12</t> 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 . 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 )
    C2c12 Myoblasts, supplied by ATCC, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Cell surface flip-flop of phosphatidylserine is critical for PIEZO1-mediated myotube formation"

    Article Title: Cell surface flip-flop of phosphatidylserine is critical for PIEZO1-mediated myotube formation

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04436-w

    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 . 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 )
    Figure Legend Snippet: 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 . 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 )

    Techniques Used: Staining, Transfection, Over Expression, Expressing, Flow Cytometry, Translocation Assay, Activity Assay, Fluorescence

    Impaired PIEZO1 activation in PS flippase-deficient myoblasts. a , b Suppression of agonist-induced PIEZO1 activation by PS flippase deficiency. a Left: Fura2 ratiometric measurements (F 340 /F 380 ) of Yoda1-induced Ca 2+ influx in WT, PIEZO1-deficient, CDC50A-deficient, and ATP11A-deficient C2C12 myoblasts. Right: quantification of Yoda1-induced Ca 2+ influx as the maximal increment of F 340 /F 380 (∆ ratio) in left. b Left: Fura2 ratiometric measurements of Yoda1-induced Ca 2+ influx in human primary myoblasts transfected with control, PIEZO1, CDC50A or ATP11A siRNA. Right: quantification of Yoda1-induced Ca 2+ influx in left. c Rescue of agonist-induced PIEZO1 activation in PS flippase-deficient myoblasts by overexpression of PS flippase complex components. Quantification of Yoda1-induced Ca 2+ influx in CDC50A-deficient (left) or ATP11A-deficient (right) C2C12 myoblasts expressing FLAG-tagged CDC50A, ATP11A, ATP11B or ATP11C. d , e Normal plasma membrane tension in PS flippase-deficient myoblasts. d Schematic model of membrane tension measurement using an optical trap. e Quantification of membrane tension in WT, CDC50A-deficient and cytochalasin D-treated WT C2C12 cells. f Schematic model showing PS flippase-mediated inward translocation of cell surface-exposed PS as a prerequisite for PIEZO1 activation. ** P < 0.01, *** P < 0.001, and **** P < 0.0001 (Student’s t -test). NS not significant, n sample number. Box and whiskers graph―line: median, box: upper and lower quartiles, whiskers: maxima and minima
    Figure Legend Snippet: Impaired PIEZO1 activation in PS flippase-deficient myoblasts. a , b Suppression of agonist-induced PIEZO1 activation by PS flippase deficiency. a Left: Fura2 ratiometric measurements (F 340 /F 380 ) of Yoda1-induced Ca 2+ influx in WT, PIEZO1-deficient, CDC50A-deficient, and ATP11A-deficient C2C12 myoblasts. Right: quantification of Yoda1-induced Ca 2+ influx as the maximal increment of F 340 /F 380 (∆ ratio) in left. b Left: Fura2 ratiometric measurements of Yoda1-induced Ca 2+ influx in human primary myoblasts transfected with control, PIEZO1, CDC50A or ATP11A siRNA. Right: quantification of Yoda1-induced Ca 2+ influx in left. c Rescue of agonist-induced PIEZO1 activation in PS flippase-deficient myoblasts by overexpression of PS flippase complex components. Quantification of Yoda1-induced Ca 2+ influx in CDC50A-deficient (left) or ATP11A-deficient (right) C2C12 myoblasts expressing FLAG-tagged CDC50A, ATP11A, ATP11B or ATP11C. d , e Normal plasma membrane tension in PS flippase-deficient myoblasts. d Schematic model of membrane tension measurement using an optical trap. e Quantification of membrane tension in WT, CDC50A-deficient and cytochalasin D-treated WT C2C12 cells. f Schematic model showing PS flippase-mediated inward translocation of cell surface-exposed PS as a prerequisite for PIEZO1 activation. ** P < 0.01, *** P < 0.001, and **** P < 0.0001 (Student’s t -test). NS not significant, n sample number. Box and whiskers graph―line: median, box: upper and lower quartiles, whiskers: maxima and minima

    Techniques Used: Activation Assay, Transfection, Over Expression, Expressing, Translocation Assay

    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 )
    Figure Legend Snippet: 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 )

    Techniques Used: Staining, Transfection, Expressing

    Suppression of PIEZO1 activation by cell surface-exposed PS. a , b Suppression of agonist-induced PIEZO1 activation by overexpression of phospholipid scramblases. a Schematic illustration showing inhibition of PIEZO1 activation by CA-TMEM16-mediated bidirectional translocation of PS in the plasma membrane. b Representative traces (left) and quantification (right) of Yoda1-induced Ca 2+ influx in HEK293 cells co-expressing PIEZO1 and CA-TMEM16F. c – e Rescue of agonist-induced PIEZO1 activation in PS flippase-deficient myoblasts by MαCD-catalyzed replacement of cell surface-exposed PS with exogenous PC. c Schematic illustration showing restoration of PIEZO1 activation by phospholipid exchange between PC-loaded MαCD and PS-exposing myoblasts. d , e Flow cytometry histograms of annexin V-GFP labelling ( d ) and quantification of Yoda1-induced Ca 2+ influx ( e ) of CDC50A-deficient C2C12 myoblasts pre-incubated with no lipid-, PC/PS mixture (3:1)- or PC-loaded MαCD. **** P < 0.0001 (Student’s t -test). NS not significant, n sample number. Box and whiskers graph-line: median, box: upper and lower quartiles, whiskers: maxima and minima
    Figure Legend Snippet: Suppression of PIEZO1 activation by cell surface-exposed PS. a , b Suppression of agonist-induced PIEZO1 activation by overexpression of phospholipid scramblases. a Schematic illustration showing inhibition of PIEZO1 activation by CA-TMEM16-mediated bidirectional translocation of PS in the plasma membrane. b Representative traces (left) and quantification (right) of Yoda1-induced Ca 2+ influx in HEK293 cells co-expressing PIEZO1 and CA-TMEM16F. c – e Rescue of agonist-induced PIEZO1 activation in PS flippase-deficient myoblasts by MαCD-catalyzed replacement of cell surface-exposed PS with exogenous PC. c Schematic illustration showing restoration of PIEZO1 activation by phospholipid exchange between PC-loaded MαCD and PS-exposing myoblasts. d , e Flow cytometry histograms of annexin V-GFP labelling ( d ) and quantification of Yoda1-induced Ca 2+ influx ( e ) of CDC50A-deficient C2C12 myoblasts pre-incubated with no lipid-, PC/PS mixture (3:1)- or PC-loaded MαCD. **** P < 0.0001 (Student’s t -test). NS not significant, n sample number. Box and whiskers graph-line: median, box: upper and lower quartiles, whiskers: maxima and minima

    Techniques Used: Activation Assay, Over Expression, Inhibition, Translocation Assay, Expressing, Flow Cytometry, Incubation

    Suppression of PIEZO1 activation by cell surface-inserted LysoPS. a Chemical structures of LysoPS, LysoPA and LysoPC. b – d Suppression of agonist-induced PIEZO1 activation by cell-surface LysoPS. b Schematic model showing suppression of PIEZO1 activation by insertion of LysoPS to the cell surface. c Representative traces (left) and quantification (right) of Yoda1-induced Ca 2+ influx in WT C2C12 myoblasts treated with vehicle, LysoPS, LysoPA or LysoPC. d Schematic model showing restoration of PIEZO1 activation by removal of cell surface-inserted LysoPS with lipid-free BSA. e Representative traces (left) and quantification (right) of Yoda1-induced Ca 2+ influx in WT C2C12 myoblasts treated with LysoPS and washed with lipid-free BSA. f , g Impairment of mechanical stimulation-induced PIEZO1 activation by cell-surface LysoPS. f Representative traces of the mechanically-activated current evoked by indentation (8 μm) using a glass probe in PIEZO1-expressing HEK293 cells before (grey, control) and during administration of 5 μM LysoPS (red), followed by removal of LysoPS with lipid-free BSA (blue). g Relative peak currents induced by mechanical stimulation before (control) and during administration of vehicle (grey), 5 μM LysoPS (red), and 5 μM LysoPC (blue) in f . * P < 0.05 and **** 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
    Figure Legend Snippet: Suppression of PIEZO1 activation by cell surface-inserted LysoPS. a Chemical structures of LysoPS, LysoPA and LysoPC. b – d Suppression of agonist-induced PIEZO1 activation by cell-surface LysoPS. b Schematic model showing suppression of PIEZO1 activation by insertion of LysoPS to the cell surface. c Representative traces (left) and quantification (right) of Yoda1-induced Ca 2+ influx in WT C2C12 myoblasts treated with vehicle, LysoPS, LysoPA or LysoPC. d Schematic model showing restoration of PIEZO1 activation by removal of cell surface-inserted LysoPS with lipid-free BSA. e Representative traces (left) and quantification (right) of Yoda1-induced Ca 2+ influx in WT C2C12 myoblasts treated with LysoPS and washed with lipid-free BSA. f , g Impairment of mechanical stimulation-induced PIEZO1 activation by cell-surface LysoPS. f Representative traces of the mechanically-activated current evoked by indentation (8 μm) using a glass probe in PIEZO1-expressing HEK293 cells before (grey, control) and during administration of 5 μM LysoPS (red), followed by removal of LysoPS with lipid-free BSA (blue). g Relative peak currents induced by mechanical stimulation before (control) and during administration of vehicle (grey), 5 μM LysoPS (red), and 5 μM LysoPC (blue) in f . * P < 0.05 and **** 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

    Techniques Used: Activation Assay, Expressing

    RhoA/ROCK-mediated actomyosin formation via the PS flippase/PIEZO1 pathway required for myotube morphology. a Schematic diagram of the PS flippase/PIEZO1 axis (black) and activators for the RhoA/ROCK/actomyosin pathway (red). b , c Suppressed cortical accumulation of phosphorylated MLC2 (P-MLC2) in PS flippase-deficient and PIEZO1-deficient myotubes. b Localization of P-MLC2 (anti-P-MLC2 antibody, green), F-actin (phalloidin, red), and nuclei (DAPI, cyan) in WT, PIEZO1-deficient, CDC50A-deficient, and ATP11A-deficient C2C12 syncytia. Intense co-localization of P-MLC2 and F-actin was observed on the cell periphery of WT myotubes. c Cortex vs. cytoplasm ratio of P-MLC2 signals in b . d , e Rescue of myotube formation by stable expression of a phospho-mimetic form of MLC2 in PIEZO1-deficient C2C12 syncytia. d Syncytia formed by WT and PIEZO1-deficient C2C12 myoblasts stably expressing WT MLC2 (MLC2-WT-GFP) or phospho-mimetic MLC2 (MLC2-DD-GFP) were visualized by immunofluorescent staining with anti-MyHC antibody (differentiated cells, red) and DAPI (nuclei, cyan). e Left: cell fusion evaluated as percentages of syncytia containing ≥50 nuclei in d . Right: polarized elongation evaluated as percentages of syncytia with aspect ratios ≥3 in d . f Rescue of myotube formation by activation of the RhoA/ROCK/actomyosin pathway in PS flippase- or PIEZO1-deficient C2C12 syncytia. Syncytia formed by WT, PIEZO1-deficient, CDC50A-deficient, and ATP11A-deficient C2C12 syncytia after treatment with calyculin A (a myosin II activator) or CN03 (a selective RhoA activator) were visualized by immunofluorescent staining with anti-MyHC antibody and DAPI. Morphologies of the syncytia are shown in Supplementary Figure . Left: cell fusion evaluated as percentages of syncytia containing ≥50 nuclei. Right: polarized elongation evaluated as percentages of syncytia with aspect ratios ≥3. **** 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: 20 μm ( b ), 100 μm ( d )
    Figure Legend Snippet: RhoA/ROCK-mediated actomyosin formation via the PS flippase/PIEZO1 pathway required for myotube morphology. a Schematic diagram of the PS flippase/PIEZO1 axis (black) and activators for the RhoA/ROCK/actomyosin pathway (red). b , c Suppressed cortical accumulation of phosphorylated MLC2 (P-MLC2) in PS flippase-deficient and PIEZO1-deficient myotubes. b Localization of P-MLC2 (anti-P-MLC2 antibody, green), F-actin (phalloidin, red), and nuclei (DAPI, cyan) in WT, PIEZO1-deficient, CDC50A-deficient, and ATP11A-deficient C2C12 syncytia. Intense co-localization of P-MLC2 and F-actin was observed on the cell periphery of WT myotubes. c Cortex vs. cytoplasm ratio of P-MLC2 signals in b . d , e Rescue of myotube formation by stable expression of a phospho-mimetic form of MLC2 in PIEZO1-deficient C2C12 syncytia. d Syncytia formed by WT and PIEZO1-deficient C2C12 myoblasts stably expressing WT MLC2 (MLC2-WT-GFP) or phospho-mimetic MLC2 (MLC2-DD-GFP) were visualized by immunofluorescent staining with anti-MyHC antibody (differentiated cells, red) and DAPI (nuclei, cyan). e Left: cell fusion evaluated as percentages of syncytia containing ≥50 nuclei in d . Right: polarized elongation evaluated as percentages of syncytia with aspect ratios ≥3 in d . f Rescue of myotube formation by activation of the RhoA/ROCK/actomyosin pathway in PS flippase- or PIEZO1-deficient C2C12 syncytia. Syncytia formed by WT, PIEZO1-deficient, CDC50A-deficient, and ATP11A-deficient C2C12 syncytia after treatment with calyculin A (a myosin II activator) or CN03 (a selective RhoA activator) were visualized by immunofluorescent staining with anti-MyHC antibody and DAPI. Morphologies of the syncytia are shown in Supplementary Figure . Left: cell fusion evaluated as percentages of syncytia containing ≥50 nuclei. Right: polarized elongation evaluated as percentages of syncytia with aspect ratios ≥3. **** 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: 20 μm ( b ), 100 μm ( d )

    Techniques Used: Expressing, Stable Transfection, Staining, Activation Assay

    c2c12 myoblasts  (ATCC)


    Bioz Verified Symbol ATCC is a verified supplier
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  • 90

    Structured Review

    ATCC c2c12 myoblasts
    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 <t>C2C12</t> 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 . 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 )
    C2c12 Myoblasts, supplied by ATCC, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/c2c12 myoblasts/product/ATCC
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    c2c12 myoblasts - by Bioz Stars, 2024-02
    90/100 stars

    Images

    1) Product Images from "Cell surface flip-flop of phosphatidylserine is critical for PIEZO1-mediated myotube formation"

    Article Title: Cell surface flip-flop of phosphatidylserine is critical for PIEZO1-mediated myotube formation

    Journal: Nature Communications

    doi: 10.1038/s41467-018-04436-w

    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 . 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 )
    Figure Legend Snippet: 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 . 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 )

    Techniques Used: Staining, Transfection, Over Expression, Expressing, Flow Cytometry, Translocation Assay, Activity Assay, Fluorescence

    Impaired PIEZO1 activation in PS flippase-deficient myoblasts. a , b Suppression of agonist-induced PIEZO1 activation by PS flippase deficiency. a Left: Fura2 ratiometric measurements (F 340 /F 380 ) of Yoda1-induced Ca 2+ influx in WT, PIEZO1-deficient, CDC50A-deficient, and ATP11A-deficient C2C12 myoblasts. Right: quantification of Yoda1-induced Ca 2+ influx as the maximal increment of F 340 /F 380 (∆ ratio) in left. b Left: Fura2 ratiometric measurements of Yoda1-induced Ca 2+ influx in human primary myoblasts transfected with control, PIEZO1, CDC50A or ATP11A siRNA. Right: quantification of Yoda1-induced Ca 2+ influx in left. c Rescue of agonist-induced PIEZO1 activation in PS flippase-deficient myoblasts by overexpression of PS flippase complex components. Quantification of Yoda1-induced Ca 2+ influx in CDC50A-deficient (left) or ATP11A-deficient (right) C2C12 myoblasts expressing FLAG-tagged CDC50A, ATP11A, ATP11B or ATP11C. d , e Normal plasma membrane tension in PS flippase-deficient myoblasts. d Schematic model of membrane tension measurement using an optical trap. e Quantification of membrane tension in WT, CDC50A-deficient and cytochalasin D-treated WT C2C12 cells. f Schematic model showing PS flippase-mediated inward translocation of cell surface-exposed PS as a prerequisite for PIEZO1 activation. ** P < 0.01, *** P < 0.001, and **** P < 0.0001 (Student’s t -test). NS not significant, n sample number. Box and whiskers graph―line: median, box: upper and lower quartiles, whiskers: maxima and minima
    Figure Legend Snippet: Impaired PIEZO1 activation in PS flippase-deficient myoblasts. a , b Suppression of agonist-induced PIEZO1 activation by PS flippase deficiency. a Left: Fura2 ratiometric measurements (F 340 /F 380 ) of Yoda1-induced Ca 2+ influx in WT, PIEZO1-deficient, CDC50A-deficient, and ATP11A-deficient C2C12 myoblasts. Right: quantification of Yoda1-induced Ca 2+ influx as the maximal increment of F 340 /F 380 (∆ ratio) in left. b Left: Fura2 ratiometric measurements of Yoda1-induced Ca 2+ influx in human primary myoblasts transfected with control, PIEZO1, CDC50A or ATP11A siRNA. Right: quantification of Yoda1-induced Ca 2+ influx in left. c Rescue of agonist-induced PIEZO1 activation in PS flippase-deficient myoblasts by overexpression of PS flippase complex components. Quantification of Yoda1-induced Ca 2+ influx in CDC50A-deficient (left) or ATP11A-deficient (right) C2C12 myoblasts expressing FLAG-tagged CDC50A, ATP11A, ATP11B or ATP11C. d , e Normal plasma membrane tension in PS flippase-deficient myoblasts. d Schematic model of membrane tension measurement using an optical trap. e Quantification of membrane tension in WT, CDC50A-deficient and cytochalasin D-treated WT C2C12 cells. f Schematic model showing PS flippase-mediated inward translocation of cell surface-exposed PS as a prerequisite for PIEZO1 activation. ** P < 0.01, *** P < 0.001, and **** P < 0.0001 (Student’s t -test). NS not significant, n sample number. Box and whiskers graph―line: median, box: upper and lower quartiles, whiskers: maxima and minima

    Techniques Used: Activation Assay, Transfection, Over Expression, Expressing, Translocation Assay

    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 )
    Figure Legend Snippet: 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 )

    Techniques Used: Staining, Transfection, Expressing

    Suppression of PIEZO1 activation by cell surface-exposed PS. a , b Suppression of agonist-induced PIEZO1 activation by overexpression of phospholipid scramblases. a Schematic illustration showing inhibition of PIEZO1 activation by CA-TMEM16-mediated bidirectional translocation of PS in the plasma membrane. b Representative traces (left) and quantification (right) of Yoda1-induced Ca 2+ influx in HEK293 cells co-expressing PIEZO1 and CA-TMEM16F. c – e Rescue of agonist-induced PIEZO1 activation in PS flippase-deficient myoblasts by MαCD-catalyzed replacement of cell surface-exposed PS with exogenous PC. c Schematic illustration showing restoration of PIEZO1 activation by phospholipid exchange between PC-loaded MαCD and PS-exposing myoblasts. d , e Flow cytometry histograms of annexin V-GFP labelling ( d ) and quantification of Yoda1-induced Ca 2+ influx ( e ) of CDC50A-deficient C2C12 myoblasts pre-incubated with no lipid-, PC/PS mixture (3:1)- or PC-loaded MαCD. **** P < 0.0001 (Student’s t -test). NS not significant, n sample number. Box and whiskers graph-line: median, box: upper and lower quartiles, whiskers: maxima and minima
    Figure Legend Snippet: Suppression of PIEZO1 activation by cell surface-exposed PS. a , b Suppression of agonist-induced PIEZO1 activation by overexpression of phospholipid scramblases. a Schematic illustration showing inhibition of PIEZO1 activation by CA-TMEM16-mediated bidirectional translocation of PS in the plasma membrane. b Representative traces (left) and quantification (right) of Yoda1-induced Ca 2+ influx in HEK293 cells co-expressing PIEZO1 and CA-TMEM16F. c – e Rescue of agonist-induced PIEZO1 activation in PS flippase-deficient myoblasts by MαCD-catalyzed replacement of cell surface-exposed PS with exogenous PC. c Schematic illustration showing restoration of PIEZO1 activation by phospholipid exchange between PC-loaded MαCD and PS-exposing myoblasts. d , e Flow cytometry histograms of annexin V-GFP labelling ( d ) and quantification of Yoda1-induced Ca 2+ influx ( e ) of CDC50A-deficient C2C12 myoblasts pre-incubated with no lipid-, PC/PS mixture (3:1)- or PC-loaded MαCD. **** P < 0.0001 (Student’s t -test). NS not significant, n sample number. Box and whiskers graph-line: median, box: upper and lower quartiles, whiskers: maxima and minima

    Techniques Used: Activation Assay, Over Expression, Inhibition, Translocation Assay, Expressing, Flow Cytometry, Incubation

    Suppression of PIEZO1 activation by cell surface-inserted LysoPS. a Chemical structures of LysoPS, LysoPA and LysoPC. b – d Suppression of agonist-induced PIEZO1 activation by cell-surface LysoPS. b Schematic model showing suppression of PIEZO1 activation by insertion of LysoPS to the cell surface. c Representative traces (left) and quantification (right) of Yoda1-induced Ca 2+ influx in WT C2C12 myoblasts treated with vehicle, LysoPS, LysoPA or LysoPC. d Schematic model showing restoration of PIEZO1 activation by removal of cell surface-inserted LysoPS with lipid-free BSA. e Representative traces (left) and quantification (right) of Yoda1-induced Ca 2+ influx in WT C2C12 myoblasts treated with LysoPS and washed with lipid-free BSA. f , g Impairment of mechanical stimulation-induced PIEZO1 activation by cell-surface LysoPS. f Representative traces of the mechanically-activated current evoked by indentation (8 μm) using a glass probe in PIEZO1-expressing HEK293 cells before (grey, control) and during administration of 5 μM LysoPS (red), followed by removal of LysoPS with lipid-free BSA (blue). g Relative peak currents induced by mechanical stimulation before (control) and during administration of vehicle (grey), 5 μM LysoPS (red), and 5 μM LysoPC (blue) in f . * P < 0.05 and **** 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
    Figure Legend Snippet: Suppression of PIEZO1 activation by cell surface-inserted LysoPS. a Chemical structures of LysoPS, LysoPA and LysoPC. b – d Suppression of agonist-induced PIEZO1 activation by cell-surface LysoPS. b Schematic model showing suppression of PIEZO1 activation by insertion of LysoPS to the cell surface. c Representative traces (left) and quantification (right) of Yoda1-induced Ca 2+ influx in WT C2C12 myoblasts treated with vehicle, LysoPS, LysoPA or LysoPC. d Schematic model showing restoration of PIEZO1 activation by removal of cell surface-inserted LysoPS with lipid-free BSA. e Representative traces (left) and quantification (right) of Yoda1-induced Ca 2+ influx in WT C2C12 myoblasts treated with LysoPS and washed with lipid-free BSA. f , g Impairment of mechanical stimulation-induced PIEZO1 activation by cell-surface LysoPS. f Representative traces of the mechanically-activated current evoked by indentation (8 μm) using a glass probe in PIEZO1-expressing HEK293 cells before (grey, control) and during administration of 5 μM LysoPS (red), followed by removal of LysoPS with lipid-free BSA (blue). g Relative peak currents induced by mechanical stimulation before (control) and during administration of vehicle (grey), 5 μM LysoPS (red), and 5 μM LysoPC (blue) in f . * P < 0.05 and **** 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

    Techniques Used: Activation Assay, Expressing

    RhoA/ROCK-mediated actomyosin formation via the PS flippase/PIEZO1 pathway required for myotube morphology. a Schematic diagram of the PS flippase/PIEZO1 axis (black) and activators for the RhoA/ROCK/actomyosin pathway (red). b , c Suppressed cortical accumulation of phosphorylated MLC2 (P-MLC2) in PS flippase-deficient and PIEZO1-deficient myotubes. b Localization of P-MLC2 (anti-P-MLC2 antibody, green), F-actin (phalloidin, red), and nuclei (DAPI, cyan) in WT, PIEZO1-deficient, CDC50A-deficient, and ATP11A-deficient C2C12 syncytia. Intense co-localization of P-MLC2 and F-actin was observed on the cell periphery of WT myotubes. c Cortex vs. cytoplasm ratio of P-MLC2 signals in b . d , e Rescue of myotube formation by stable expression of a phospho-mimetic form of MLC2 in PIEZO1-deficient C2C12 syncytia. d Syncytia formed by WT and PIEZO1-deficient C2C12 myoblasts stably expressing WT MLC2 (MLC2-WT-GFP) or phospho-mimetic MLC2 (MLC2-DD-GFP) were visualized by immunofluorescent staining with anti-MyHC antibody (differentiated cells, red) and DAPI (nuclei, cyan). e Left: cell fusion evaluated as percentages of syncytia containing ≥50 nuclei in d . Right: polarized elongation evaluated as percentages of syncytia with aspect ratios ≥3 in d . f Rescue of myotube formation by activation of the RhoA/ROCK/actomyosin pathway in PS flippase- or PIEZO1-deficient C2C12 syncytia. Syncytia formed by WT, PIEZO1-deficient, CDC50A-deficient, and ATP11A-deficient C2C12 syncytia after treatment with calyculin A (a myosin II activator) or CN03 (a selective RhoA activator) were visualized by immunofluorescent staining with anti-MyHC antibody and DAPI. Morphologies of the syncytia are shown in Supplementary Figure . Left: cell fusion evaluated as percentages of syncytia containing ≥50 nuclei. Right: polarized elongation evaluated as percentages of syncytia with aspect ratios ≥3. **** 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: 20 μm ( b ), 100 μm ( d )
    Figure Legend Snippet: RhoA/ROCK-mediated actomyosin formation via the PS flippase/PIEZO1 pathway required for myotube morphology. a Schematic diagram of the PS flippase/PIEZO1 axis (black) and activators for the RhoA/ROCK/actomyosin pathway (red). b , c Suppressed cortical accumulation of phosphorylated MLC2 (P-MLC2) in PS flippase-deficient and PIEZO1-deficient myotubes. b Localization of P-MLC2 (anti-P-MLC2 antibody, green), F-actin (phalloidin, red), and nuclei (DAPI, cyan) in WT, PIEZO1-deficient, CDC50A-deficient, and ATP11A-deficient C2C12 syncytia. Intense co-localization of P-MLC2 and F-actin was observed on the cell periphery of WT myotubes. c Cortex vs. cytoplasm ratio of P-MLC2 signals in b . d , e Rescue of myotube formation by stable expression of a phospho-mimetic form of MLC2 in PIEZO1-deficient C2C12 syncytia. d Syncytia formed by WT and PIEZO1-deficient C2C12 myoblasts stably expressing WT MLC2 (MLC2-WT-GFP) or phospho-mimetic MLC2 (MLC2-DD-GFP) were visualized by immunofluorescent staining with anti-MyHC antibody (differentiated cells, red) and DAPI (nuclei, cyan). e Left: cell fusion evaluated as percentages of syncytia containing ≥50 nuclei in d . Right: polarized elongation evaluated as percentages of syncytia with aspect ratios ≥3 in d . f Rescue of myotube formation by activation of the RhoA/ROCK/actomyosin pathway in PS flippase- or PIEZO1-deficient C2C12 syncytia. Syncytia formed by WT, PIEZO1-deficient, CDC50A-deficient, and ATP11A-deficient C2C12 syncytia after treatment with calyculin A (a myosin II activator) or CN03 (a selective RhoA activator) were visualized by immunofluorescent staining with anti-MyHC antibody and DAPI. Morphologies of the syncytia are shown in Supplementary Figure . Left: cell fusion evaluated as percentages of syncytia containing ≥50 nuclei. Right: polarized elongation evaluated as percentages of syncytia with aspect ratios ≥3. **** 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: 20 μm ( b ), 100 μm ( d )

    Techniques Used: Expressing, Stable Transfection, Staining, Activation Assay

    brevibacterium epidermidis  (ATCC)


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    ATCC brevibacterium epidermidis
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    1) Product Images from "Direct Growth of Bacteria in Headspace Vials Allows for Screening of Volatiles by Gas Chromatography Mass Spectrometry"

    Article Title: Direct Growth of Bacteria in Headspace Vials Allows for Screening of Volatiles by Gas Chromatography Mass Spectrometry

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.00491

    Bacterial strains and ordering information.
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    brevibacterium epidermidis atcc 49089  (ATCC)


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    b epidermidis atcc 49089  (ATCC)


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    ATCC c2c12 myoblasts
    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 <t>C2C12</t> 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 . 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 )
    C2c12 Myoblasts, supplied by ATCC, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC brevibacterium epidermidis
    Bacterial strains and ordering information.
    Brevibacterium Epidermidis, supplied by ATCC, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC brevibacterium epidermidis atcc 49089
    Bacterial strains and ordering information.
    Brevibacterium Epidermidis Atcc 49089, supplied by ATCC, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC b epidermidis atcc 49089
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    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 . 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 )

    Journal: Nature Communications

    Article Title: Cell surface flip-flop of phosphatidylserine is critical for PIEZO1-mediated myotube formation

    doi: 10.1038/s41467-018-04436-w

    Figure Lengend Snippet: 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 . 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 )

    Article Snippet: The other mouse cDNA clones were obtained from mouse (C57BL/6J, Japan SLC Inc.) brain or C2C12 myoblasts (ATCC). pcDNA3.1-Clover-mRuby2 (#49089), mPIEZO1-IRES-eGFP (#80925), pX260 (#42229), and pX330 (#42230) were purchased from Addgene. pGFP-AHD was generated according to previous literature . pX330-PGKpuro was generated by inserting a PGK promoter and a puromycin-resistant gene (from pX260) into pX330.

    Techniques: Staining, Transfection, Over Expression, Expressing, Flow Cytometry, Translocation Assay, Activity Assay, Fluorescence

    Impaired PIEZO1 activation in PS flippase-deficient myoblasts. a , b Suppression of agonist-induced PIEZO1 activation by PS flippase deficiency. a Left: Fura2 ratiometric measurements (F 340 /F 380 ) of Yoda1-induced Ca 2+ influx in WT, PIEZO1-deficient, CDC50A-deficient, and ATP11A-deficient C2C12 myoblasts. Right: quantification of Yoda1-induced Ca 2+ influx as the maximal increment of F 340 /F 380 (∆ ratio) in left. b Left: Fura2 ratiometric measurements of Yoda1-induced Ca 2+ influx in human primary myoblasts transfected with control, PIEZO1, CDC50A or ATP11A siRNA. Right: quantification of Yoda1-induced Ca 2+ influx in left. c Rescue of agonist-induced PIEZO1 activation in PS flippase-deficient myoblasts by overexpression of PS flippase complex components. Quantification of Yoda1-induced Ca 2+ influx in CDC50A-deficient (left) or ATP11A-deficient (right) C2C12 myoblasts expressing FLAG-tagged CDC50A, ATP11A, ATP11B or ATP11C. d , e Normal plasma membrane tension in PS flippase-deficient myoblasts. d Schematic model of membrane tension measurement using an optical trap. e Quantification of membrane tension in WT, CDC50A-deficient and cytochalasin D-treated WT C2C12 cells. f Schematic model showing PS flippase-mediated inward translocation of cell surface-exposed PS as a prerequisite for PIEZO1 activation. ** P < 0.01, *** P < 0.001, and **** P < 0.0001 (Student’s t -test). NS not significant, n sample number. Box and whiskers graph―line: median, box: upper and lower quartiles, whiskers: maxima and minima

    Journal: Nature Communications

    Article Title: Cell surface flip-flop of phosphatidylserine is critical for PIEZO1-mediated myotube formation

    doi: 10.1038/s41467-018-04436-w

    Figure Lengend Snippet: Impaired PIEZO1 activation in PS flippase-deficient myoblasts. a , b Suppression of agonist-induced PIEZO1 activation by PS flippase deficiency. a Left: Fura2 ratiometric measurements (F 340 /F 380 ) of Yoda1-induced Ca 2+ influx in WT, PIEZO1-deficient, CDC50A-deficient, and ATP11A-deficient C2C12 myoblasts. Right: quantification of Yoda1-induced Ca 2+ influx as the maximal increment of F 340 /F 380 (∆ ratio) in left. b Left: Fura2 ratiometric measurements of Yoda1-induced Ca 2+ influx in human primary myoblasts transfected with control, PIEZO1, CDC50A or ATP11A siRNA. Right: quantification of Yoda1-induced Ca 2+ influx in left. c Rescue of agonist-induced PIEZO1 activation in PS flippase-deficient myoblasts by overexpression of PS flippase complex components. Quantification of Yoda1-induced Ca 2+ influx in CDC50A-deficient (left) or ATP11A-deficient (right) C2C12 myoblasts expressing FLAG-tagged CDC50A, ATP11A, ATP11B or ATP11C. d , e Normal plasma membrane tension in PS flippase-deficient myoblasts. d Schematic model of membrane tension measurement using an optical trap. e Quantification of membrane tension in WT, CDC50A-deficient and cytochalasin D-treated WT C2C12 cells. f Schematic model showing PS flippase-mediated inward translocation of cell surface-exposed PS as a prerequisite for PIEZO1 activation. ** P < 0.01, *** P < 0.001, and **** P < 0.0001 (Student’s t -test). NS not significant, n sample number. Box and whiskers graph―line: median, box: upper and lower quartiles, whiskers: maxima and minima

    Article Snippet: The other mouse cDNA clones were obtained from mouse (C57BL/6J, Japan SLC Inc.) brain or C2C12 myoblasts (ATCC). pcDNA3.1-Clover-mRuby2 (#49089), mPIEZO1-IRES-eGFP (#80925), pX260 (#42229), and pX330 (#42230) were purchased from Addgene. pGFP-AHD was generated according to previous literature . pX330-PGKpuro was generated by inserting a PGK promoter and a puromycin-resistant gene (from pX260) into pX330.

    Techniques: Activation Assay, Transfection, Over Expression, Expressing, Translocation Assay

    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 )

    Journal: Nature Communications

    Article Title: Cell surface flip-flop of phosphatidylserine is critical for PIEZO1-mediated myotube formation

    doi: 10.1038/s41467-018-04436-w

    Figure Lengend Snippet: 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 )

    Article Snippet: The other mouse cDNA clones were obtained from mouse (C57BL/6J, Japan SLC Inc.) brain or C2C12 myoblasts (ATCC). pcDNA3.1-Clover-mRuby2 (#49089), mPIEZO1-IRES-eGFP (#80925), pX260 (#42229), and pX330 (#42230) were purchased from Addgene. pGFP-AHD was generated according to previous literature . pX330-PGKpuro was generated by inserting a PGK promoter and a puromycin-resistant gene (from pX260) into pX330.

    Techniques: Staining, Transfection, Expressing

    Suppression of PIEZO1 activation by cell surface-exposed PS. a , b Suppression of agonist-induced PIEZO1 activation by overexpression of phospholipid scramblases. a Schematic illustration showing inhibition of PIEZO1 activation by CA-TMEM16-mediated bidirectional translocation of PS in the plasma membrane. b Representative traces (left) and quantification (right) of Yoda1-induced Ca 2+ influx in HEK293 cells co-expressing PIEZO1 and CA-TMEM16F. c – e Rescue of agonist-induced PIEZO1 activation in PS flippase-deficient myoblasts by MαCD-catalyzed replacement of cell surface-exposed PS with exogenous PC. c Schematic illustration showing restoration of PIEZO1 activation by phospholipid exchange between PC-loaded MαCD and PS-exposing myoblasts. d , e Flow cytometry histograms of annexin V-GFP labelling ( d ) and quantification of Yoda1-induced Ca 2+ influx ( e ) of CDC50A-deficient C2C12 myoblasts pre-incubated with no lipid-, PC/PS mixture (3:1)- or PC-loaded MαCD. **** P < 0.0001 (Student’s t -test). NS not significant, n sample number. Box and whiskers graph-line: median, box: upper and lower quartiles, whiskers: maxima and minima

    Journal: Nature Communications

    Article Title: Cell surface flip-flop of phosphatidylserine is critical for PIEZO1-mediated myotube formation

    doi: 10.1038/s41467-018-04436-w

    Figure Lengend Snippet: Suppression of PIEZO1 activation by cell surface-exposed PS. a , b Suppression of agonist-induced PIEZO1 activation by overexpression of phospholipid scramblases. a Schematic illustration showing inhibition of PIEZO1 activation by CA-TMEM16-mediated bidirectional translocation of PS in the plasma membrane. b Representative traces (left) and quantification (right) of Yoda1-induced Ca 2+ influx in HEK293 cells co-expressing PIEZO1 and CA-TMEM16F. c – e Rescue of agonist-induced PIEZO1 activation in PS flippase-deficient myoblasts by MαCD-catalyzed replacement of cell surface-exposed PS with exogenous PC. c Schematic illustration showing restoration of PIEZO1 activation by phospholipid exchange between PC-loaded MαCD and PS-exposing myoblasts. d , e Flow cytometry histograms of annexin V-GFP labelling ( d ) and quantification of Yoda1-induced Ca 2+ influx ( e ) of CDC50A-deficient C2C12 myoblasts pre-incubated with no lipid-, PC/PS mixture (3:1)- or PC-loaded MαCD. **** P < 0.0001 (Student’s t -test). NS not significant, n sample number. Box and whiskers graph-line: median, box: upper and lower quartiles, whiskers: maxima and minima

    Article Snippet: The other mouse cDNA clones were obtained from mouse (C57BL/6J, Japan SLC Inc.) brain or C2C12 myoblasts (ATCC). pcDNA3.1-Clover-mRuby2 (#49089), mPIEZO1-IRES-eGFP (#80925), pX260 (#42229), and pX330 (#42230) were purchased from Addgene. pGFP-AHD was generated according to previous literature . pX330-PGKpuro was generated by inserting a PGK promoter and a puromycin-resistant gene (from pX260) into pX330.

    Techniques: Activation Assay, Over Expression, Inhibition, Translocation Assay, Expressing, Flow Cytometry, Incubation

    Suppression of PIEZO1 activation by cell surface-inserted LysoPS. a Chemical structures of LysoPS, LysoPA and LysoPC. b – d Suppression of agonist-induced PIEZO1 activation by cell-surface LysoPS. b Schematic model showing suppression of PIEZO1 activation by insertion of LysoPS to the cell surface. c Representative traces (left) and quantification (right) of Yoda1-induced Ca 2+ influx in WT C2C12 myoblasts treated with vehicle, LysoPS, LysoPA or LysoPC. d Schematic model showing restoration of PIEZO1 activation by removal of cell surface-inserted LysoPS with lipid-free BSA. e Representative traces (left) and quantification (right) of Yoda1-induced Ca 2+ influx in WT C2C12 myoblasts treated with LysoPS and washed with lipid-free BSA. f , g Impairment of mechanical stimulation-induced PIEZO1 activation by cell-surface LysoPS. f Representative traces of the mechanically-activated current evoked by indentation (8 μm) using a glass probe in PIEZO1-expressing HEK293 cells before (grey, control) and during administration of 5 μM LysoPS (red), followed by removal of LysoPS with lipid-free BSA (blue). g Relative peak currents induced by mechanical stimulation before (control) and during administration of vehicle (grey), 5 μM LysoPS (red), and 5 μM LysoPC (blue) in f . * P < 0.05 and **** 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

    Journal: Nature Communications

    Article Title: Cell surface flip-flop of phosphatidylserine is critical for PIEZO1-mediated myotube formation

    doi: 10.1038/s41467-018-04436-w

    Figure Lengend Snippet: Suppression of PIEZO1 activation by cell surface-inserted LysoPS. a Chemical structures of LysoPS, LysoPA and LysoPC. b – d Suppression of agonist-induced PIEZO1 activation by cell-surface LysoPS. b Schematic model showing suppression of PIEZO1 activation by insertion of LysoPS to the cell surface. c Representative traces (left) and quantification (right) of Yoda1-induced Ca 2+ influx in WT C2C12 myoblasts treated with vehicle, LysoPS, LysoPA or LysoPC. d Schematic model showing restoration of PIEZO1 activation by removal of cell surface-inserted LysoPS with lipid-free BSA. e Representative traces (left) and quantification (right) of Yoda1-induced Ca 2+ influx in WT C2C12 myoblasts treated with LysoPS and washed with lipid-free BSA. f , g Impairment of mechanical stimulation-induced PIEZO1 activation by cell-surface LysoPS. f Representative traces of the mechanically-activated current evoked by indentation (8 μm) using a glass probe in PIEZO1-expressing HEK293 cells before (grey, control) and during administration of 5 μM LysoPS (red), followed by removal of LysoPS with lipid-free BSA (blue). g Relative peak currents induced by mechanical stimulation before (control) and during administration of vehicle (grey), 5 μM LysoPS (red), and 5 μM LysoPC (blue) in f . * P < 0.05 and **** 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

    Article Snippet: The other mouse cDNA clones were obtained from mouse (C57BL/6J, Japan SLC Inc.) brain or C2C12 myoblasts (ATCC). pcDNA3.1-Clover-mRuby2 (#49089), mPIEZO1-IRES-eGFP (#80925), pX260 (#42229), and pX330 (#42230) were purchased from Addgene. pGFP-AHD was generated according to previous literature . pX330-PGKpuro was generated by inserting a PGK promoter and a puromycin-resistant gene (from pX260) into pX330.

    Techniques: Activation Assay, Expressing

    RhoA/ROCK-mediated actomyosin formation via the PS flippase/PIEZO1 pathway required for myotube morphology. a Schematic diagram of the PS flippase/PIEZO1 axis (black) and activators for the RhoA/ROCK/actomyosin pathway (red). b , c Suppressed cortical accumulation of phosphorylated MLC2 (P-MLC2) in PS flippase-deficient and PIEZO1-deficient myotubes. b Localization of P-MLC2 (anti-P-MLC2 antibody, green), F-actin (phalloidin, red), and nuclei (DAPI, cyan) in WT, PIEZO1-deficient, CDC50A-deficient, and ATP11A-deficient C2C12 syncytia. Intense co-localization of P-MLC2 and F-actin was observed on the cell periphery of WT myotubes. c Cortex vs. cytoplasm ratio of P-MLC2 signals in b . d , e Rescue of myotube formation by stable expression of a phospho-mimetic form of MLC2 in PIEZO1-deficient C2C12 syncytia. d Syncytia formed by WT and PIEZO1-deficient C2C12 myoblasts stably expressing WT MLC2 (MLC2-WT-GFP) or phospho-mimetic MLC2 (MLC2-DD-GFP) were visualized by immunofluorescent staining with anti-MyHC antibody (differentiated cells, red) and DAPI (nuclei, cyan). e Left: cell fusion evaluated as percentages of syncytia containing ≥50 nuclei in d . Right: polarized elongation evaluated as percentages of syncytia with aspect ratios ≥3 in d . f Rescue of myotube formation by activation of the RhoA/ROCK/actomyosin pathway in PS flippase- or PIEZO1-deficient C2C12 syncytia. Syncytia formed by WT, PIEZO1-deficient, CDC50A-deficient, and ATP11A-deficient C2C12 syncytia after treatment with calyculin A (a myosin II activator) or CN03 (a selective RhoA activator) were visualized by immunofluorescent staining with anti-MyHC antibody and DAPI. Morphologies of the syncytia are shown in Supplementary Figure . Left: cell fusion evaluated as percentages of syncytia containing ≥50 nuclei. Right: polarized elongation evaluated as percentages of syncytia with aspect ratios ≥3. **** 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: 20 μm ( b ), 100 μm ( d )

    Journal: Nature Communications

    Article Title: Cell surface flip-flop of phosphatidylserine is critical for PIEZO1-mediated myotube formation

    doi: 10.1038/s41467-018-04436-w

    Figure Lengend Snippet: RhoA/ROCK-mediated actomyosin formation via the PS flippase/PIEZO1 pathway required for myotube morphology. a Schematic diagram of the PS flippase/PIEZO1 axis (black) and activators for the RhoA/ROCK/actomyosin pathway (red). b , c Suppressed cortical accumulation of phosphorylated MLC2 (P-MLC2) in PS flippase-deficient and PIEZO1-deficient myotubes. b Localization of P-MLC2 (anti-P-MLC2 antibody, green), F-actin (phalloidin, red), and nuclei (DAPI, cyan) in WT, PIEZO1-deficient, CDC50A-deficient, and ATP11A-deficient C2C12 syncytia. Intense co-localization of P-MLC2 and F-actin was observed on the cell periphery of WT myotubes. c Cortex vs. cytoplasm ratio of P-MLC2 signals in b . d , e Rescue of myotube formation by stable expression of a phospho-mimetic form of MLC2 in PIEZO1-deficient C2C12 syncytia. d Syncytia formed by WT and PIEZO1-deficient C2C12 myoblasts stably expressing WT MLC2 (MLC2-WT-GFP) or phospho-mimetic MLC2 (MLC2-DD-GFP) were visualized by immunofluorescent staining with anti-MyHC antibody (differentiated cells, red) and DAPI (nuclei, cyan). e Left: cell fusion evaluated as percentages of syncytia containing ≥50 nuclei in d . Right: polarized elongation evaluated as percentages of syncytia with aspect ratios ≥3 in d . f Rescue of myotube formation by activation of the RhoA/ROCK/actomyosin pathway in PS flippase- or PIEZO1-deficient C2C12 syncytia. Syncytia formed by WT, PIEZO1-deficient, CDC50A-deficient, and ATP11A-deficient C2C12 syncytia after treatment with calyculin A (a myosin II activator) or CN03 (a selective RhoA activator) were visualized by immunofluorescent staining with anti-MyHC antibody and DAPI. Morphologies of the syncytia are shown in Supplementary Figure . Left: cell fusion evaluated as percentages of syncytia containing ≥50 nuclei. Right: polarized elongation evaluated as percentages of syncytia with aspect ratios ≥3. **** 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: 20 μm ( b ), 100 μm ( d )

    Article Snippet: The other mouse cDNA clones were obtained from mouse (C57BL/6J, Japan SLC Inc.) brain or C2C12 myoblasts (ATCC). pcDNA3.1-Clover-mRuby2 (#49089), mPIEZO1-IRES-eGFP (#80925), pX260 (#42229), and pX330 (#42230) were purchased from Addgene. pGFP-AHD was generated according to previous literature . pX330-PGKpuro was generated by inserting a PGK promoter and a puromycin-resistant gene (from pX260) into pX330.

    Techniques: Expressing, Stable Transfection, Staining, Activation Assay

    Bacterial strains and ordering information.

    Journal: Frontiers in Microbiology

    Article Title: Direct Growth of Bacteria in Headspace Vials Allows for Screening of Volatiles by Gas Chromatography Mass Spectrometry

    doi: 10.3389/fmicb.2018.00491

    Figure Lengend Snippet: Bacterial strains and ordering information.

    Article Snippet: Brevibacterium epidermidis , NCDO 2286 , ATCC , ATCC 35514.

    Techniques: