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 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).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).