Fig. 1
PAK1 regulates actin core dynamics and invadopodia disassembly. MDA-MB-231 cells were either untreated, or lentiviral infected with control or PAK1 shRNA to generate stable knockdowns, or treated with 1 µM IPA-3. a Representative Western blot of PAK1 levels in MDA-MB-231, control and PAK1 shRNA cells. b Quantification of Western blot experiments in. Means ± S.E. (error bars) from three independent experiments are shown. Student’s t-test was used to determine significant differences. Different letters denote a value significantly different from MDA-MB-231 cells (p < 0.05). c, d Cells were plated on gelatin-coated coverslips for 3 h, fixed, permeabilized, and stained using anti-TKS5 antibody, followed by Alexa594-conjugated secondary antibody and Alexa488-phalloidin to stain F-actin. d Single confocal slices of the ventral surface of cells are shown. Areas of colocalization are seen in the overlay as yellow. c Quantification of TKS5 and F-actin-containing punctae were counted using confocal microscope. Means ± SEM from 3 independent experiments in which 20–30 cells per sample were assessed are shown. A two way ANOVA comparing all four groups showed no significance (n.s.). e–g Cells were plated on Alexa594-labeled gelatin for 5 h. GM6001 was added for 3 h, washed out and cells were fixed 2 h later. e Invadopodium-based degradation of the 594-gelatin matrix of individual cells was quantified using ImageJ software. Means ± SEM from 3 independent experiments in which 10–20 cells per sample were measured are shown. A two way ANOVA was used to determine significant differences. Different letters denote significant differences (p < 0.05). f Actin core size at invadopodium-based degradation sites of individual cells was quantified using ImageJ software. g Confocal microscopy images at the ventral cell surface showing spots of invadopodium degradation (black holes) with overlaying actin cores (green). Means ± SEM from 3 independent experiments in which 10–20 cells per sample were measured are shown. One-way ANOVA was used to determine significance. Different letters denote significant differences (p < 0.05). Scale bar = 10 µm
