Finally, we performed TUNEL assays and FACS analysis, measuring proportions of apoptotic cells, to investigate whether MELK is involved in the apoptosis cascade through the Bcl-GL-related pathway.
TUNEL assays and FACS analysis revealed that overexpression of wild-type MELK suppressed Bcl-GL-induced apoptosis, while that of D150A-MELK did not.
For FACS analysis, the cells were harvested by trypsinization and fixed with 70% ethanol at room temperature for 30 minutes. After centrifuging to remove the ethanol, the cells were treated with 0.5 ml of PBS(-) containing 1 mg/ml of RNase I (Sigma-Aldrich) for 30 minutes followed by staining in 1 ml of PBS(-) containing 20 mg/ml of propidium iodide (Sigma-Aldrich) for 30 minutes. The cells selected from at least 10,000 ungated cells were analyzed for DNA content by flow cytometry (FACS calibur; Becton Dickinson, San Diego, CA, USA). The data were analyzed using CELLQuest software (FACS calibur; Becton Dickinson). Assays were done in triplicate independently.
To investigate this hypothesis, we transiently co-transfected two plasmid clones designed to express HA-tagged MELK (WT or D150A) and Flag-tagged Bcl-GL into COS7 cells, and then performed a TUNEL assay and FACS analysis to measure the proportions of apoptotic cells (see Material and methods).
As shown in Figure 5d, FACS analysis of the cells under the same conditions also confirmed that the overexpression of Bcl-GL increased the sub-G1 population of cells compared with the mock-transfected cells.
As reported previously [14], we demonstrated by TUNEL assay and FACS analysis that introduction of full-length Bcl-GL into COS7 cells induced apoptosis. However, under the same conditions, addition of exogenous WT-MELK suppressed induction of apoptosis by Bcl-GL, but addition of D150A-MELK did not (Figure 5b–d).
(d) FACS analysis of cells collected after transfection with pCAGGSnHC (HA-Mock), pCAGGSn3FH (Flag-Mock), HA-tagged MELK (WT and D150A), Flag-tagged Bcl-GL expression vectors, and combinations of these.Notably, we did not detect any cell-cycle-dependent alteration of MDC1 S329/T331 phosphorylation relative to total MDC1 protein content by using flow cytometry, which suggests that SDTD phosphorylation occurs throughout interphase (supplementary Fig S1B online).(A) Altered steady-state levels of superoxide as shown by increased oxidation of DHE in HCT116 SIRT3 overexpressing cells. Control (pcDNA vector), wild-type SIRT3 (wt-SIRT3), or a deacetylation SIRT3 mutant (mut-SIRT3) cell lines were analyzed by flow cytometry for the amount of hydrolyzed DHE per 10,000 cells represented as Mean Florescent Intensity (MFI).