Figure 3

BMI1 represses the BMP pathway and controls cell migration of primary Group 4 MB cells and MB cell lines. (A) Analysis of BMI1 protein expression in various MB cell lines. (B) Effective BMI1 knock down is seen in DAOY and D-458 48 hrs after treatment with BMI1 siRNA, as compared to siScr and Untreated (Untr) controls. (C) Increased phosphorylation of SMAD1,5,8 proteins (pSMAD1,5,8) as compared to total SMAD1,5,8 is seen in DAOY cells following BMI1 shRNA treatment (shBMI1) as compared to shScr. (D) BMI1 protein expression is seen in primary Group 4 MB cells (ICb-1299), and the expression levels are comparable to that of DAOY. (E) An effective BMI1 knock down is achieved in ICb-1299 primary cells following BMI1 shRNA treatment. (F) Increased pSMAD1,5,8 expression as compared to total SMAD1,5,8 is also seen in ICb-1299 upon BMI1 knock down, indicative of BMP pathway activation. (G) Schematic of organotypic cerebellar slice co-culture used as an ex vivo assay to study MB cell migration. The DAOY or ICb-1299 cell spheres are placed on the slices and allowed to migrate for 8 days. (H) Confocal images at day 8 demonstrate migration of GFP labelled DAOY cells on the cerebellar slice. Reduced cell migration in shBMI1 (right) compared to shScr treated (left) DAOY cells. (I and J) Decreased area of migration (I) and a decreased distance of migration (J) is seen in both DAOY and ICb-1299 cells treated with shBMI1, compared to shScr controls. Three areas were assessed on each slice and a total of three slices were analysed in each group (N = 3). Error bars in graphs represent SD. *, P < 0.05; **, P < 0.01. Scale bar is 250 μm. Abbreviations: GFP, Green Fluorescent Protein; DAPI, 4′,6-diamidino-2-phenylindole.