Figure 4

Knocking down BCAR3 promotes transforming growth factor β–induced cell migration. (a) SCP2 cells were transfected, scratched and treated as described in the Methods section and subjected to real-time migration assays. The images are computer-generated pictures showing positions of cell boundaries at the beginning of the assay (time 0) and at the end of the assay (48 hours). The black regions represent occupancy of cells after initial scratching, the white regions represent empty space not filled by cells (the scratch) and the gray regions represent initial scratch areas filled by cells at the end of the assay (migration). The images are representative of three independent experiments. Because of space limitations, the images for small interfering RNA (siRNA) #2 are not shown. (b) Migration profiles over time were compared for mock-transfected cells and cells transfected with two different BCAR3 siRNAs. Error bars show standard error of the mean of six biological replicates in one experiment. (c) and (d) MDA-MB-231 cells were transfected, scratched using a P200 tip and stimulated with or without 200 pM transforming growth factor β (TGFβ). Cells were subjected to real-time cell migration assays using the IncuCyte automated imaging system. Images obtained at 0, 24 and 36 hours were analyzed using TScratch to automatically recognize wound area and calculate the percentage of the fields of view occupied by cells. The results show cell migration expressed as increases in area percentage at 24 hours and 36 hours (c) and fold induction by TGFβ at 36 hours (d), respectively. The percentages of the fields of view were quantified from ten image sets of a representative experiment (n = 3). Error bars show standard error of the mean. An asterisk indicates statistical significance (*P < 0.05) as determined by unpaired Student’s t-test.