Figure 7

Src kinase inhibitor suppresses accumulation of p120 and organization of microrafts. (A) Ric10 cells were cultured for 24 h in pmDM and then further cultured in pmDM supplemented with 0.1% DMSO (a-h) or SU6656 (i-p) for further 9 h. The cells were fixed and subjected to immunostaining for p120 (green) and p120PY228 (red). Cell nuclei were stained with DAPI (blue). Images were obtained by phase contrast and epifluorescence microscopy. Arrowheads represent membrane ruffles (e-h). Arrows represent cell contacts (m-p). Scale bars: 100 μm (a-d, i-l), 25 μm (e-h, m-p). (B) Ric10 cells were cultured in pmDM for 24 h and sequentially observed under phase contrast microscopy by time-lapse recording with 2.5 min interval (upper row). Then, the medium was switched to pmDM supplemented with SU6656 (10 μM). The same field was sequentially observed approximately 10 minutes after administration (second row). 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 (third row). Then the medium was switched to pmDM supplemented with both 100 μM vanadate and 10 μM SU6656 and sequentially observed by time-lapse recording (lowest row). Arrows represent the leading edge of lamellipodium. Arrowheads represent membrane ruffles. (C) GGS25 cells were cultured for 24 h in pmDM and then further cultured for 3 h in pmDM supplemented with 0.1% DMSO (upper row) or 10 μM SU6656 (lower row). The cells were sequentially observed at 1 min intervals under epifluorescence microscopy by time-lapse recording. Arrows represent microrafts where GFP-GPI was accumulated.