Figure 5.

MELK involvement in the apoptosis cascade through Bcl-G_L. HA (hemagglutinin)-tagged MELK (HA-wild-type (WT)-MELK or HA-kinase-dead (D150A)-MELK) and Flag-tagged Bcl-G_L (Flag-Bcl-G_L) expression vectors were co-transfected into COS7 cells for 24 hours. (a) The expression of MELK and Bcl-G_L proteins in the co-transfected cells were examined by western blot analysis. (b) TUNEL assays after transfection with pCAGGSnHC (HA-Mock), pCAGGSn3FH (Flag-Mock), HA-tagged MELK (WT and D150A), Flag-tagged Bcl-G_L expression vectors, and combinations of these. Apoptotic cells were measured by counting of TUNEL staining (means ± standard deviation, n = 3; P = 0.0001; unpaired t-test). (c) Representative images of TUNEL assays. Cells were labeled with DAPI (4',6-diamidino-2-phenylindole) for counting of total cell number. Apoptotic cells with DNA strand breaks were labeled with green fluorescence. (d) FACS analysis of cells collected after transfection with pCAGGSnHC (HA-Mock), pCAGGSn3FH (Flag-Mock), HA-tagged MELK (WT and D150A), Flag-tagged Bcl-G_L expression vectors, and combinations of these. Proportions of apoptotic cells are indicated as percentages of sub-G1 populations. Each value represents the average of three experiments (means ± standard deviation, n = 3).