Figure 2

KDM1A inhibition impairs cell proliferation and migration and induces apoptosis in human medulloblastoma cell lines. a Bars represent KDM1A expression measured using real-time RT-PCR and normalized to the geometric mean of GAPDH, UBC and HPRT expression in DAOY and ONS-76 cell lines 72 h after KDM1A knockdown or mock transfection. ***p < 0.0001 b Knockdown of KDM1A protein was confirmed by western blotting of whole-cell lysates from DAOY and ONS-76 cells. β-actin served as loading control. c The DAOY and ONS-76 medulloblastoma cell lines were transfected with siRNA directed against KDMA1, and cell viability was measured using the MTT assay. Extinction relative to mock-transfected cultures at 72 h is shown. ***p < 0.0001 d Proliferation of DAOY and ONS-76 cells following mock transfection or transfection with siRNA directed against KDM1A was assessed by BrdU ELISA. Bars show extinction relative to mock-transfected cultures at 72 h. ***p < 0.0001 e Apoptosis in DAOY and ONS-76 cells was measured by Cell Death Detection ELISA™ 72 h after transfection with either siRNA directed against KDMA1 or mock transfection. Extinction is relative to mock-transfected cultures. ***p < 0.0001, *p < 0.05 f Migratory activity was assessed for the ONS-76 cell line 48 h after transfection with either siRNA directed against KDM1A or mock transfection in Boyden chamber assays. Representative images of DAPI-stained mock-transfected control cells (ONS-76 ctrl) and KDM1A-knockdown cells (ONS-76 siKDM1A) invading the membrane (scale bars = 100 μm). g Statistical analysis of results from Boyden chamber assays 24 h after DAOY and ONS-76 cells, either transfected with siRNA directed against KDM1A or mock-transfected, were plated in the upper chamber. Bars display quantity of cells per mm square which migrated through the membrane. **p < 0.01, *p < 0.05.