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Open Access Research article

Regulation of pre-fusion events: recruitment of M-cadherin to microrafts organized at fusion-competent sites of myogenic cells

Atsushi Mukai and Naohiro Hashimoto*

Author Affiliations

Department of Regenerative Medicine, National Institute for Longevity Sciences, National Center for Geriatrics and Gerontology, 35 Gengo, Morioka, Oobu, Aichi 474-8522, Japan

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BMC Cell Biology 2013, 14:37  doi:10.1186/1471-2121-14-37

Published: 27 August 2013

Additional files

Additional file 1:

Rac1 inhibitor suppresses microraft organization during myogenic differentiation. GGS25 cells were cultured in pmGM. GFP-GPI was sequentially observed at 1 min intervals under epifluorescence microscopy by time-lapse recording. Many microrafts were organized in pmDM, whereas the frequency of microraft organization and the signal intensity of GFP-GPI declined in pmGM and pmDM with NSC23766.

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Additional file 2:

GGS25 cells were cultured in pmDM for up to 37 h. GFP-GPI was sequentially observed at 1 min intervals under epifluorescence microscopy by time-lapse recording. Many microrafts were organized in pmDM.

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Additional file 3:

GGS25 cells were cultured in pmDM with NSC23766 (100 μM) for up to 37 h. GFP-GPI was sequentially observed at 1 min intervals under epifluorescence microscopy by time-lapse recording. The frequency of microraft organization and the signal intensity of GFP-GPI declined in pmDM with NSC23766.

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Additional file 4:

Effects of Src kinase inhibitor or protein tyrosine phosphatase inhibitor vanadate on fusion–related proteins of myogenic cells. Ric10 cells were cultured for 24 h in pmDM and then cultured in pmDM supplemented with 0.1% DMSO (−) or SU6656 (+) for 9 h (A), or vanadate (1, 10, 100 μM) for 24 h (B). Total lysates (20 μg of proteins) were subjected to immunoblot analyses. MyHC, myosin heavy chain; p120PY228, tyrosine phosphorylated p120; p120PT310, threonine phosphorylated p120. β-tubulin was used as a loading control.

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Additional file 5:

Src kinase inhibitor doesn’t suppress accumulation of p120 at cell contacts. Ric10 cells were cultured for 24 h in pmDM and then cultured in pmDM supplemented with 0.1% DMSO (Ctrl) or SU6656 (100 μM) for 24 h. Tyrosine-phosphorylated p120 accumulated at cell-cell contacts in both control cultures (Ctrl) and SU6656- (green) and p120PY228 (red)-treated cultures. Cell nuclei were stained with DAPI (blue). Images were obtained by epifluorescence microscopy.

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Additional file 6:

Vanadate antagonizes the inhibitory effect of Src kinase inhibitor on membrane ruffling. Ric10 cells were cultured in pmDM for 24 h and sequentially observed under phase contrast microscopy by time-lapse recording.Images were recorded every 2.5 min by phase-contrast time-lapse microscopy. Membrane ruffling in pmDM (Additional file 6) was suppressed in pmDM supplemented with SU6656 (Additional file 7). Membrane ruffling in pmDM supplemented with vanadate (Additional file 8) was not suppressed in pmDM supplemented with SU6656 and vanadate (Additional file 9).

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Additional file 7:

Ric10 cells were cultured in pmDM for 24 h and sequentially observed under phase contrast microscopy by time-lapse recording (Additional file6). Then, the medium was switched to pmDM supplemented with SU6656 (10 μM). The same field was sequentially observed approximately 10 minutes after administration. Images were recorded every 2.5 min by phase-contrast time-lapse microscopy. Membrane rufflingwas suppressed in pmDM supplemented with SU6656.

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Additional file 8:

Ric10 cells were cultured for 24 h in pmDM and then incubated for 20 min in pmDM supplemented with 100 μM vanadate. The cells were sequentially observed by time-lapse recording. Images were recorded every 2.5 min by phase-contrast time-lapse microscopy. Membrane ruffling in pmDM supplemented with.

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Additional file 9:

Ric10 cells were cultured for 24 h in pmDM and then incubated for 20 min in pmDM supplemented with 100 μM vanadate. The cells were sequentially observed by time-lapse recording (Additional file 8). Then the medium was switched to pmDM supplemented with both 100 μM vanadate and 10 μM SU6656 and sequentially observed by time-lapse recording. Images were recorded every 2.5 min by phase-contrast time-lapse microscopy. Membrane ruffling in pmDM supplemented with vanadate (Additional file 8) was not suppressed in pmDM supplemented with SU6656 and vanadate.

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Additional file 10:

Src kinase inhibitor suppresses organization of microrafts. GGS25 cells were cultured for 24 h in pmDM and then further cultured for 3 h in pmDM supplemented with 0.1% DMSO or 10 μM SU6656. Microrafts appeared as white spots and disappeared in control cultures (Additional file 10), whereas SU6656 prevented microraft organization and any plasma membrane movement (Additional file 11). Nothing moved in the latter file.

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Additional file 11:

GGS25 cells were cultured for 24 h in pmDM and then further cultured for 3 h in pmDM supplemented with 10 μM SU6656. Images were recorded every min for 20 minutes by epifluorescence time-lapse microscopy. SU6656 prevented microraft organization and any plasma membrane movement. Nothing moved in the present movie.

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