MARCH5-dependent degradation of MCL1/NOXA complexes defines susceptibility to antimitotic drug treatment

摘要

Parental HeLaS3 (WT) and two clonal HeLaS3 MARCH5-KO lines created with two different small guide RNAs targeting MARCH5 (MARCH5-KO#1 and -KO#2) were treated with ABT737, etoposide (Eto), or staurosporine (STS) for 24 h. For the UV-R treatment cells were subjected to 2 mJ of UV-R and then incubated for 24 h. Cells were then harvested and propidium iodide uptake was used to measure cell death by flow cytometry. All data displayed are mean ± s.d. from five independent experiments, indicated as dots. Two-way ANOVA was used with Holm–Sidak’s multiple comparisons test to compare the WT as the control group against the two MARCH-KO lines for each treatment. All comparisons not indicated are statistically not significant (  > 0.05). Parental HeLaS3 and HeLaS3 MARCH5-KO cells were transfected with either control siRNA targeting luciferase (GL2), NOXA and GL2 siRNA (siNOXA), MCL1 and GL2 siRNA (siMCL1) or NOXA and MCL1 siRNA (DKD) for 48 h. Cells were then harvested and prepared for immunoblot analysis. The full length (fl) and the cleaved (cl) form of caspase-3 are shown. Parental HeLaS3 and two independent HeLaS3 MARCH5-KO clones were treated with Cycloheximide (CHX), harvested after the indicated time points and prepared for immunoblot analysis. Numbers below the blots show the quantification of the respective bands. Quantification was normalized to the GAPDH signal and to the untreated sample (0) of the respective genotype.