Figure 7
BMP2-induced PI3K signalling is important for directional cell migration. (A) C2C12 cell wound closure upon stimulation with 10 nM BMP2 for 14 hours compared to unstimulated control. Lipophilic carbocyanine-dye labelled cells (DiO) are presented in black pseudo-colour. Scale bar represents 200 μm. Left panel, effect of p55γ knock-down (si-p55γ) on C2C12 cell wound closure compared to scrambled transfected (si-scr) control. (B) Bar diagram depicting the intensity translocation values for three independent biological replicates for experiments shown in A using a selective mask filter (for detailed description see Methods and Additional file 7). Error bars represent standard deviation from three independent experiments. P-values from one-way analysis of variance with post-hoc Bonferroni-test are indicated. (C) Trajectories visualising the migration of p55γ knock-down (red) and scrambled control (green) C2C12 cells during BMP2-induced wound closure over a period of 12 hours. Scale bar represents 100 μm. Left panel, equal amounts of si-p55γ (red) and si-scr (green) transfected cells were labelled with DiI or DiO respectively prior to mixing and seeding. Bar diagram (right panel) summarises the intensity translocation ratios of p55γ knock-down (red) compared to scrambled control (green) C2C12 cells of 19 replicates analysed with P <0.005 considered statistically significant. (D) Transwell assay of C2C12 cells, transfected with either si-p55γ or si-LL5β compared to si-scr-transfected C2C12 cells. Cells migrated through an 8 μm porous filter upon stimulation with 10 nM BMP2 for 6 hours in the presence of 0.2% fetal calf serum. The number of cells migrated through the porous filter was counted. Bar diagram represents cell number counts per optical field. Error bars represent standard deviation from three independent experiments. P-values from one-way analysis of variance with post-hoc Bonferroni test are indicated.