Figure 5
HGF promotes cortical actin dynamics in medulloblastoma cells. (A) Immunofluorescence analysis (IFA) of c-Met and phosphorylated c-Met (p-c-Met) localization in lamellipodia of DAOY cells. Color overlay and inverted grey-scale images of p-c-Met (red), actin (green) and c-Met (blue) are shown. Magnifications are 4× of boxed area in overlay. Arrows indicate c-Met-rich lamellipodia. (B) IFA of Alexa-488-phalloidin-stained F-actin cytoskeletons in un-stimulated and HGF-stimulated (20 ng/mL, t = 10 min) DAOY cells, −/+ PHA-665752 (500 nM). Inverted grey-scale images of Alexa-488-phalloidin fluorescence are shown. Magnifications are 4× of boxed areas. Lower left magnification is 4× of sheet-like protrusion in b). Arrows: filopodia, arrowheads: leading edge of F-actin sheet (extension zone, see schematic). (C) F-actin dynamics in DAOY cells transfected with LA-mCherry and either enhanced green fluorescent protein-tagged, wild-type (wt) or kinase-defective (k/d) MAP4K4 were recorded by confocal live cell microscopy imaging. See Additional file 5: Figure S5 for still images of representative cells. Dot blots show protrusion lengths in control cells or cells expressing either EGFP-MAP4K4-wt or EGFP-MAP4K4-k/d. (D) Still images of time-lapse movies of DAOY-LA-EGFP-shScr or DAOY-LA-EGFP-shMAP4K4_1 cells stimulated with HGF (20 ng/mL). T0 is 0 h and T18 is 18 h after HGF stimulation. Inverted grey-scale of LA-EGFP fluorescence (F-actin cytoskeleton) is shown. (E) Cells were treated as described in (D). Box plots of areas in pixels covered by individual cells quantified at T0 and T18. (F) Box plot of speeds of single sh control or shMAP4K4 cells in the presence of HGF. Statistical analysis: T-test (*: P = 0.0208).