Fig. 2: A chromatin-associated pool of PKCε is cleaved by Caspase-7 in mitosis. | Nature Communications

Fig. 2: A chromatin-associated pool of PKCε is cleaved by Caspase-7 in mitosis.

From: The Aurora B specificity switch is required to protect from non-disjunction at the metaphase/anaphase transition

Fig. 2

a Pre-extraction of cells with 0.1% Triton X-100 prior to fixation revealed a chromatin-associated pool of PKCε. Images are representative of three independent experiments. Scale bar = 5 μm. b Triton X-100 insoluble fraction of asynchronous or nocodazole (500 nM) arrested DLD1 cell lysates were probed for PKCε, GFP, and Histone H3. Western blot is representative of three independent experiments. c Schematic of PKCε domains and cleavage products with putative cleavages sites described by Basu et al.12 marked by red arrows. d Immunoblot of GFP-PKCε to assess cleavage after knockdown of Caspases 3 and 7. Graph denotes the mean ± SD of three independent experiments where the proportion of cleaved 43 kDa fragment of PKCε present in the immunoblots was determined by densitometry (two-tailed Students’ t test; **P = 0.01; siControl 20.85 ± 0.76, SiCas3 13.9 ± 6.38, siCas7 7.4 ± 4.95, siCas3 + siCas7 8.98 ± 4.37). DLD1 cells were depleted of Caspase-7 and induced to express (e) GFP-PKCε wild type or (f) GFP-PKCε kinase domain and assessed for their response to metaphase catenation. Cells were treated with ±1μM ICRF193 immediately prior to time-lapse imaging and scored for the time to progress from metaphase to anaphase. Violin plots represent all cells scored, the median is in red. A minimum of 100 cells per condition in three independent experiments were analysed. (One-way ANOVA, ****P = ≤0.0001). Caspase-7 knockdown was confirmed by immunoblot for each experiment.

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