Figure 5

Cell migration of MB cell lines is regulated by BMI1 in a BMP pathway dependent fashion in vitro. (A) DAOY and D-458 cultures show an increased number of cells (in %) expressing pSMAD1,5,8 and an increased number of cohesive cell clusters upon siRNA BMI1 treatment (bottom panel) compared to siRNA Scr treatment (top panel). (B) Quantification of the number of cells expressing pSMAD1,5,8 in cultures with or without Noggin treatment (N = 3). (C) Quantification of cohesive cell clusters per HPF with or without Noggin treatment (N = 3). (D) Transwell migration assay shows a decreased migration of DAOY cells in shRNA BMI1 cultures compared to shRNA Scr, but when shBMI1 cultures are concomitantly treated with Noggin, cell migration is rescued (N = 3). (E) Gap closure assay shows similar findings. (F) Western blot demonstrating BMP pathway activation by means of increased pSMAD1,5,8 expression as compared to total SMAD1,5,8 in DAOY treated with BMP-4, compared to untreated cells. (G) Transwell migration assay shows decreased migration of DAOY cells upon BMP-4 treatment similarly to shRNA BMI1 knock down but no additive effect is seen when BMP4 treatment is applied concomitantly to shRNA BMI1 knock down (N = 4). Scale bar = 250 μm. Error bars in graphs represent SD. *, P < 0.05; **, P < 0.01. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole.