Figure 3. BMI-1 promotes the anchorage independent of ESFT cells.
A.) Phase contrast images of A4573 cells growing in standard culture conditions. siRNA-mediated knockdown of BMI-1 (siBMI1) causes A4573 cells to flatten while lipofectamine-treated and control transfected (siNS) cells continue to grow as 3-dimensional clusters.
B.) Stable knockdown of BMI-1 (shBMI1B) impairs colony formation of ESFT cells in soft agar. Images are of macroscopic colonies on the day at which control cell (shNS) media was depleted for each of the respective 3 cell lines. Each well is representative of 6 replicate wells for each condition. Similar results were obtained with a second BMI-1-targeted shRNA construct (see Supplementary Data Figure 4).
C.) ESFT cells transduced to over-express BMI-1 (pBp-BMI1) have a reduced time to macroscopic colony formation and increased colony number in soft agar when compared with control, empty-vector-transduced cells (pBp-V). Images are of macroscopic colonies on the day at which pBp-BMI1 media was depleted for each of the respective 3 cell lines. Each well is representative of 6 replicate wells for each condition.