Journal: Biochimica et biophysica acta

Article Title: Membrane repair of human skeletal muscle cells requires Annexin-A5.

doi: 10.1016/j.bbamcr.2016.06.003

Figure Lengend Snippet: Fig. 2. AnxA5-deficient myotubes present a severe defect of membrane repair. (A) The cellular content of AnxA5 and GAPDH (loading control) in LHCN myoblasts (MB) and myotubes (MT) was quantified using western-blot analysis. (B) Subcellular distribution and relative expression of endogenous AnxA5 (white) in wild-type (control) and AnxA5-targetting shRNA transduced (A5-shRNA) LHCN myotubes were analyzed by immunocytofluorescence. In the control image, the white square surrounds the magnified area displayed in the top left of the image. Scale bars = 100 μm. (C) Sequence of representative images showing the response of an AnxA5-deficient myotube to 110-mW infrared laser irradiation. Myotubes were prepared and experimented as described in the legend of Fig. 1C. Scale bar = 20 μm. (D) FM1–43 fluorescence intensity integrated over whole cell sections, averaged for about 100 cells (+/−SD). For myotubes transduced with scrambled shRNA (ctl-shRNA, empty circles) lentiviral particles, the fluorescence intensity reached a plateau within about 70 s. Instead, transduction with AnxA5-shRNA (A5-shRNA, filled circles) lentiviral particles led to a continuous increase of the fluorescence intensity.

Article Snippet: Except for caveolin-3 detection (20 min, 60 V), semi-dry electrophoretic transfer (Bio-Rad, Hercules, CA, USA) onto PVDF membrane was performed for 1 h at 100 V. The cellular content of dysferlin (230 kDa), caveolin-3 (20 kDa), AnxA1 (37 kDa), AnxA2 (36 kDa), AnxA5 (35 kDa), αtubulin (loading control, 52 kDa) and GAPDH (loading control, 37 kDa) was detected with mouse anti-dysferlin monoclonal antibody (NCL-Hamlet, Leica Biosystems, Wetzlar, Germany), mouse anticaveolin-3 monoclonal antibody (C-2, Santa Cruz Biotechnology, Heidelberg, Germany), rabbit anti-AnxA1 polyclonal antibody (PA1006, BosterBio, Pleasanton, CA, USA), mouse anti-AnxA2 monoclonal antibody (3E8-B6, Sigma, Saint-Louis, MO, USA), mouse anti-AnxA5 monoclonal antibody (AN5, Sigma), mouse anti-α-tubulin monoclonal antibody (B-1-5-2, Sigma) and rabbit anti-GAPDH polyclonal antibody (FL-335, Santa Cruz Biotechnology), respectively.

Techniques: Membrane, Control, Western Blot, Expressing, shRNA, Sequencing, Irradiation, Transduction

Journal: Biochimica et biophysica acta

Article Title: Membrane repair of human skeletal muscle cells requires Annexin-A5.

doi: 10.1016/j.bbamcr.2016.06.003

Figure Lengend Snippet: Fig. 3. Extracellular addition of recombinant AnxA5, but not AnxA5 mutant, rescues membrane repair in AnxA5-deficient myotubes. (A–B) Sequences of representative images showing the response of an AnxA5-deficient myotube (AnxA5-def. MT) to laser injury, in the presence of 10 μg/mL recombinant AnxA5 (A) or mtT-AnxA5 (B). Myotubes were prepared and experimented as described in the legend of Fig. 1C. (C) FM1–43 fluorescence intensity integrated over whole cell sections, averaged for about 100 cells (+/−SD). For myotubes transduced with AnxA5-shRNA lentiviral particles, the fluorescence intensity reached a plateau within about 70 s in the presence of 10 μg/mL recombinant AnxA5 (empty circles). Instead, the fluorescence intensity increased continuously in the presence of mtT-AnxA5 (black filled circles). Scale bars = 20 μm.

Article Snippet: Except for caveolin-3 detection (20 min, 60 V), semi-dry electrophoretic transfer (Bio-Rad, Hercules, CA, USA) onto PVDF membrane was performed for 1 h at 100 V. The cellular content of dysferlin (230 kDa), caveolin-3 (20 kDa), AnxA1 (37 kDa), AnxA2 (36 kDa), AnxA5 (35 kDa), αtubulin (loading control, 52 kDa) and GAPDH (loading control, 37 kDa) was detected with mouse anti-dysferlin monoclonal antibody (NCL-Hamlet, Leica Biosystems, Wetzlar, Germany), mouse anticaveolin-3 monoclonal antibody (C-2, Santa Cruz Biotechnology, Heidelberg, Germany), rabbit anti-AnxA1 polyclonal antibody (PA1006, BosterBio, Pleasanton, CA, USA), mouse anti-AnxA2 monoclonal antibody (3E8-B6, Sigma, Saint-Louis, MO, USA), mouse anti-AnxA5 monoclonal antibody (AN5, Sigma), mouse anti-α-tubulin monoclonal antibody (B-1-5-2, Sigma) and rabbit anti-GAPDH polyclonal antibody (FL-335, Santa Cruz Biotechnology), respectively.

Techniques: Recombinant, Mutagenesis, Membrane, Transduction, shRNA

Journal: Biochimica et biophysica acta

Article Title: Membrane repair of human skeletal muscle cells requires Annexin-A5.

doi: 10.1016/j.bbamcr.2016.06.003

Figure Lengend Snippet: Fig. 4. Subcellular trafficking of GFP-AnxA5 during sarcolemma damage in myotubes. Sequence of representative images showing the response of a GFP-AnxA5 expressing myotube to 110-mW infrared laser irradiation, in the presence of 1 mM Ca2+. GFP-AnxA5 (green) was imaged before (−1.6 s) and after laser irradiation for about 2.5 min. Red and white arrows indicate the disruption site before and after laser irradiation, respectively. Time corresponds to the period after laser irradiation. Scale bar = 20 μm.

Article Snippet: Except for caveolin-3 detection (20 min, 60 V), semi-dry electrophoretic transfer (Bio-Rad, Hercules, CA, USA) onto PVDF membrane was performed for 1 h at 100 V. The cellular content of dysferlin (230 kDa), caveolin-3 (20 kDa), AnxA1 (37 kDa), AnxA2 (36 kDa), AnxA5 (35 kDa), αtubulin (loading control, 52 kDa) and GAPDH (loading control, 37 kDa) was detected with mouse anti-dysferlin monoclonal antibody (NCL-Hamlet, Leica Biosystems, Wetzlar, Germany), mouse anticaveolin-3 monoclonal antibody (C-2, Santa Cruz Biotechnology, Heidelberg, Germany), rabbit anti-AnxA1 polyclonal antibody (PA1006, BosterBio, Pleasanton, CA, USA), mouse anti-AnxA2 monoclonal antibody (3E8-B6, Sigma, Saint-Louis, MO, USA), mouse anti-AnxA5 monoclonal antibody (AN5, Sigma), mouse anti-α-tubulin monoclonal antibody (B-1-5-2, Sigma) and rabbit anti-GAPDH polyclonal antibody (FL-335, Santa Cruz Biotechnology), respectively.

Techniques: Sequencing, Expressing, Irradiation, Disruption

Journal: Biochimica et biophysica acta

Article Title: Membrane repair of human skeletal muscle cells requires Annexin-A5.

doi: 10.1016/j.bbamcr.2016.06.003

Figure Lengend Snippet: Fig. 5. Binding of extracellular AnxA5 to the disrupted site of laser-injured myotubes. LHCN myotubes were submitted to 110-mW laser-induced membrane rupture in the presence of 3 μg/mL Cy5-AnxA5 (white) diluted in D-PBS containing 1 mM Ca2+. Before laser injury (−1.6 s), simultaneous recording in bright field microscopy (frame 1) and fluorescence microscopy (frame 2) was performed. Area of membrane irradiation is marked with red and white arrow before and after irradiation, respectively. Image frames 3, 4 and 5 were recorded 6.4 s, 64 s and 146 s after irradiation, respectively. Scale bar = 20 μm.

Article Snippet: Except for caveolin-3 detection (20 min, 60 V), semi-dry electrophoretic transfer (Bio-Rad, Hercules, CA, USA) onto PVDF membrane was performed for 1 h at 100 V. The cellular content of dysferlin (230 kDa), caveolin-3 (20 kDa), AnxA1 (37 kDa), AnxA2 (36 kDa), AnxA5 (35 kDa), αtubulin (loading control, 52 kDa) and GAPDH (loading control, 37 kDa) was detected with mouse anti-dysferlin monoclonal antibody (NCL-Hamlet, Leica Biosystems, Wetzlar, Germany), mouse anticaveolin-3 monoclonal antibody (C-2, Santa Cruz Biotechnology, Heidelberg, Germany), rabbit anti-AnxA1 polyclonal antibody (PA1006, BosterBio, Pleasanton, CA, USA), mouse anti-AnxA2 monoclonal antibody (3E8-B6, Sigma, Saint-Louis, MO, USA), mouse anti-AnxA5 monoclonal antibody (AN5, Sigma), mouse anti-α-tubulin monoclonal antibody (B-1-5-2, Sigma) and rabbit anti-GAPDH polyclonal antibody (FL-335, Santa Cruz Biotechnology), respectively.

Techniques: Binding Assay, Membrane, Microscopy, Irradiation

Journal: Biochimica et biophysica acta

Article Title: Membrane repair of human skeletal muscle cells requires Annexin-A5.

doi: 10.1016/j.bbamcr.2016.06.003

Figure Lengend Snippet: Fig. 7. Distribution of endogenous AnxA5 at the disruption site analyzed by immunoelectron microscopy. Representative images of a myotube irradiated and immunostained for AnxA5. After irradiation, myotubes were fixed with 1% glutaraldehyde for 20 min at room temperature and then immunostained for AnxA5 using a secondary antibody conjugated to both Alexa Fluor 488 and a gold nanoparticle (1.4 nm), enabling to combine fluorescence (A) and TEM (C–D) imaging. Time interval between laser injury and cell fixation was 5 min. (A) By fluorescence microscopy, an accumulation of endogenous AnxA5 (white) was observed at the disruption site (arrow). Scale bar = 20 μm. (B) After cell embedding, two serial sections of the damaged area (S.1–S.2) were collected. The red arrow indicates the direction of laser irradiation. (C–D) TEM images of the two sections described in B. Red numbers indicate the position of three vesicles, for sake of clarity, on each image. AnxA5 (black particles) was present at the surface of lipid vesicles and at the edges of the torn membrane (black arrowheads). Scale bars = 200 nm.

Article Snippet: Except for caveolin-3 detection (20 min, 60 V), semi-dry electrophoretic transfer (Bio-Rad, Hercules, CA, USA) onto PVDF membrane was performed for 1 h at 100 V. The cellular content of dysferlin (230 kDa), caveolin-3 (20 kDa), AnxA1 (37 kDa), AnxA2 (36 kDa), AnxA5 (35 kDa), αtubulin (loading control, 52 kDa) and GAPDH (loading control, 37 kDa) was detected with mouse anti-dysferlin monoclonal antibody (NCL-Hamlet, Leica Biosystems, Wetzlar, Germany), mouse anticaveolin-3 monoclonal antibody (C-2, Santa Cruz Biotechnology, Heidelberg, Germany), rabbit anti-AnxA1 polyclonal antibody (PA1006, BosterBio, Pleasanton, CA, USA), mouse anti-AnxA2 monoclonal antibody (3E8-B6, Sigma, Saint-Louis, MO, USA), mouse anti-AnxA5 monoclonal antibody (AN5, Sigma), mouse anti-α-tubulin monoclonal antibody (B-1-5-2, Sigma) and rabbit anti-GAPDH polyclonal antibody (FL-335, Santa Cruz Biotechnology), respectively.

Techniques: Disruption, Immuno-Electron Microscopy, Irradiation, Imaging, Microscopy, Membrane

Journal: Biochimica et biophysica acta

Article Title: Membrane repair of human skeletal muscle cells requires Annexin-A5.

doi: 10.1016/j.bbamcr.2016.06.003

Figure Lengend Snippet: Fig. 6. Subcellular localization of endogenous AnxA5 in damaged myotubes. Myotubes were irradiated with a 110-mW infrared laser (white arrows) in D-PBS containing 1 mM Ca2+, fixed either 5 min (A–B) or 25 min (C–D) after irradiation and immunostained for AnxA5. 5 min after laser injury, 85% of myotubes exhibited an accumulation of AnxA5 at the disruption site (A), while remaining 15% showed no accumulation (B). 25 min after laser injury, 30% of myotubes exhibited an accumulation of AnxA5 (C), while remaining 70% did not (D). Scale bars = 20 μm.

Article Snippet: Except for caveolin-3 detection (20 min, 60 V), semi-dry electrophoretic transfer (Bio-Rad, Hercules, CA, USA) onto PVDF membrane was performed for 1 h at 100 V. The cellular content of dysferlin (230 kDa), caveolin-3 (20 kDa), AnxA1 (37 kDa), AnxA2 (36 kDa), AnxA5 (35 kDa), αtubulin (loading control, 52 kDa) and GAPDH (loading control, 37 kDa) was detected with mouse anti-dysferlin monoclonal antibody (NCL-Hamlet, Leica Biosystems, Wetzlar, Germany), mouse anticaveolin-3 monoclonal antibody (C-2, Santa Cruz Biotechnology, Heidelberg, Germany), rabbit anti-AnxA1 polyclonal antibody (PA1006, BosterBio, Pleasanton, CA, USA), mouse anti-AnxA2 monoclonal antibody (3E8-B6, Sigma, Saint-Louis, MO, USA), mouse anti-AnxA5 monoclonal antibody (AN5, Sigma), mouse anti-α-tubulin monoclonal antibody (B-1-5-2, Sigma) and rabbit anti-GAPDH polyclonal antibody (FL-335, Santa Cruz Biotechnology), respectively.

Techniques: Irradiation, Disruption

Journal: Biochimica et biophysica acta

Article Title: Membrane repair of human skeletal muscle cells requires Annexin-A5.

doi: 10.1016/j.bbamcr.2016.06.003

Figure Lengend Snippet: Fig. 9. AnxA5 accumulates at the disruption site of damaged L578 myotubes. (A) The cellular content of AnxA5 and GAPDH (loading control) in L578 myoblasts (MB) and myotubes (MT) was quantified using western-blot analysis. (B) Subcellular localization of endogenous AnxA5 in intact L578 myotubes was analyzed by immunocytofluorescence. Scale bar = 20 μm. (C) Subcellular distribution of endogenous AnxA5 in a damaged L578 myotube. Myotubes were irradiated with a 110-mW infrared laser (white arrow) in D-PBS containing 1 mM Ca2+, fixed 5 min after irradiation and immunostained for AnxA5 (white). Scale bar = 20 μm. (D) Binding of extracellular AnxA5 to the disrupted site of laser-injured myotubes. L578 myotubes were treated and analyzed as described in the legend of the Fig. 5. Image frames 3, 4 and 5 were recorded 6.4 s, 51.2 s and 71.2 s after irradiation, respectively. Scale bar = 10 μm.

Article Snippet: Except for caveolin-3 detection (20 min, 60 V), semi-dry electrophoretic transfer (Bio-Rad, Hercules, CA, USA) onto PVDF membrane was performed for 1 h at 100 V. The cellular content of dysferlin (230 kDa), caveolin-3 (20 kDa), AnxA1 (37 kDa), AnxA2 (36 kDa), AnxA5 (35 kDa), αtubulin (loading control, 52 kDa) and GAPDH (loading control, 37 kDa) was detected with mouse anti-dysferlin monoclonal antibody (NCL-Hamlet, Leica Biosystems, Wetzlar, Germany), mouse anticaveolin-3 monoclonal antibody (C-2, Santa Cruz Biotechnology, Heidelberg, Germany), rabbit anti-AnxA1 polyclonal antibody (PA1006, BosterBio, Pleasanton, CA, USA), mouse anti-AnxA2 monoclonal antibody (3E8-B6, Sigma, Saint-Louis, MO, USA), mouse anti-AnxA5 monoclonal antibody (AN5, Sigma), mouse anti-α-tubulin monoclonal antibody (B-1-5-2, Sigma) and rabbit anti-GAPDH polyclonal antibody (FL-335, Santa Cruz Biotechnology), respectively.

Techniques: Disruption, Control, Western Blot, Irradiation, Binding Assay

Journal: Biochimica et biophysica acta

Article Title: Membrane repair of human skeletal muscle cells requires Annexin-A5.

doi: 10.1016/j.bbamcr.2016.06.003

Figure Lengend Snippet: Fig. 10. TEM analysis of the ultrastructure of the disruption site and the distribution of endogenous AnxA5 in LHCN and L578 myotubes. (A–B) Representative images of LHCN (A) and L578 myotubes (B) irradiated and immunostained for AnxA5 as described in the legend of Fig. 7. By fluorescence microscopy (left-hand panels), an accumulation of endogenous AnxA5 (white) was observed at the disruption site (arrow). Scale bars = 20 μm. After cell embedding, sections of the damaged cells were observed by TEM (right-hand panels). In TEM image from (B), the white squares surround the magnified area displayed in (C). Scale bars = 1 μm. (C) Three magnified areas from (B) showing the presence of AnxA5 (black particles) at the surface of lipid vesicles (arrows) and at the edges of the torn membrane (arrowheads). Scale bars = 1 μm.

Article Snippet: Except for caveolin-3 detection (20 min, 60 V), semi-dry electrophoretic transfer (Bio-Rad, Hercules, CA, USA) onto PVDF membrane was performed for 1 h at 100 V. The cellular content of dysferlin (230 kDa), caveolin-3 (20 kDa), AnxA1 (37 kDa), AnxA2 (36 kDa), AnxA5 (35 kDa), αtubulin (loading control, 52 kDa) and GAPDH (loading control, 37 kDa) was detected with mouse anti-dysferlin monoclonal antibody (NCL-Hamlet, Leica Biosystems, Wetzlar, Germany), mouse anticaveolin-3 monoclonal antibody (C-2, Santa Cruz Biotechnology, Heidelberg, Germany), rabbit anti-AnxA1 polyclonal antibody (PA1006, BosterBio, Pleasanton, CA, USA), mouse anti-AnxA2 monoclonal antibody (3E8-B6, Sigma, Saint-Louis, MO, USA), mouse anti-AnxA5 monoclonal antibody (AN5, Sigma), mouse anti-α-tubulin monoclonal antibody (B-1-5-2, Sigma) and rabbit anti-GAPDH polyclonal antibody (FL-335, Santa Cruz Biotechnology), respectively.

Techniques: Disruption, Irradiation, Microscopy, Membrane