Figure 5: NOXA controls the mitotic lifespan by promoting MCL1 degradation.

(a) HeLaS3 cells were synchronized and treated as described in Fig. 3 and processed for immunoblotting to evaluate NOXA expression. (b) Cells were transfected with the indicated siRNAs and either left untreated (Asynch.) or synchronized by double-thymidine block and harvested after the release in G2. Cells were analysed by immunoblotting (c) Fate profiles of individual HeLaS3-H2B–GFP cells subjected to treatment with DMSO, Nocodazole or BI2536 after transfection with the indicated siRNAs. Time in hours is indicated (d) Box (interquartile range) and whisker (min to max) plots showing the elapsed time (min) between nuclear envelope breakdown (NEBD) and cell death for individual cells traced in c. The fraction (X/50 events) of cells undergoing the fate of interest is indicated. **P<0.01; ***P<0.001; ****P<0.0001 using Mann–Whitney test. (e) HeLaS3 cells were transfected with the indicated siRNAs, synchronized by double-thymidine block, harvested after increasing times of mitotic arrest, triggered by Nocodazole and processed for immunoblot with the indicated antibodies. (f) Parental HeLa-FlpIN cells or transgenic for either NOXA-WT or NOXA-L29E were transfected with the indicated siRNAs, treated with Staurosporine (STS), left asynchronous (Async) or synchronized by double-thymidine block and released into medium containing Nocodozole and 20 or 100 ng ml⁻¹ doxocycline (DXC). Cells were harvested in G2 or after increasing times of mitotic arrest and processed for immunoblot with the indicated antibodies. NS, not significant.