Figure 5

TNK2 controls and BCAR1 control breast cancer cell invasion via distinct mechanisms. (a) Invasion assays were carried out using MDA-MB-231 cells transfected with targeting siRNA (S1, S3) and nontargeting SiRNA (N). At 48 hours post transfection, cells were detached, counted, added to modified Boyden chambers coated with Matrigel, and allowed to invade over a period of 48 hours. Following this, cells that had invaded were fixed in situ, stained and counted. The assay was repeated a total of six times, revealing an average inhibition of invasion of 85.5%, P < 0.0001 (S1) and 91.2%, P < 0.0001 (S3) relative to the nontargeting SiRNA control (N). A similar effect on the invasive capacity of MDA-MB-231 cells was seen in cells treated with SiRNA targeting breast cancer antioestrogen resistance 1 (BCAR1) silencing, where the average inhibition of invasion was 52.5%, P = 0.0628 (#2) and 93%, P < 0.0001 (#3). A representative image is shown illustrating the cells that had invaded the Matrigel over the course of the invasion assay. (b) Effect of targeting TNK2 SiRNA (S1) versus BCAR1 SiRNA (#3) and nontargeting SiRNA (N) on the levels of cell surface epidermal growth factor receptors (EGFRs) as measured by fluorescence-activated cell sorting analysis. On average, a 35% reduction of cell surface receptors can be seen at timepoint 0 (P = 0.0037) in cells treated with SiRNA targeting TNK2 (S1); however, the reduction seen with BCAR1 was not significant (P = 0.156).(c) Effect of TNK2 siRNA treatment on the total cellular EGFR expression as measured by western blot analysis. Approximately a 10% reduction in total EGFR is observed with TNK2 siRNA treatment. There is no statistically significant reduction seen with BCAR1 siRNA treatment. A representative western blot illustrating the downregulation of TNK2 (S1) and BCAR1 (#3) achieved by SiRNA treatment and their respective EGFR levels relative to β-actin is shown. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.