Induced exit from mitosis in NRVM is accompanied by rapid reassembly of α-actinin and titin.
(A) After 16 hr of nocodazole exposure to enrich cells at prometaphase, fresh media alone (control) or with 9 μM Ro3306 replaced nocodazole-containing media. For each time point and treatment condition, 3000 NRVM were counted in the low magnification (10X) images and absolute numbers of cells in each mitotic sub-phase were plotted. (B) Immunofluorescence of a single representative cell chosen from each of: interphase cells after thymidine treatment, nocodazole enriched prometaphase cells, anaphase cells 1 hr after nocodazole release and prophase-like cells (indicated by arrows) after 60 min Ro3306 treatment following nocodazole release, are shown for both titin (M8) and α-actinin. (C) The α-actinin disassembly index was assessed for the different cell groups as shown above in (A) and (B). Analyzed cells were randomly chosen from three independent experiments. Data are presented as the median in a box and whisker plot. One way ANOVA followed by Tukey's multiple comparison test was performed. * p < 0.01 between control and Ro3306 groups is highlighted.