Fig. 2

Interaction of both Borealin and INCENP with microtubules is important for error free mitosis. a Schematic of siBorealin-mediated knockdown rescue experiment. b Representative frames from time-lapse imaging of SiR-DNA labeled cells treated with Borealin siRNA and rescued with expression of indicated Borealin transgene, form one of three independent replicates. The quantitation is shown in d and e. c Western blots of cells expressing indicated transgene and treated as in a with Borealin or control siRNA, showing endogenous Borealin and LAP-Borealin expression. Tubulin staining is used as loading control. The lysates were collected 8–9 h after the second thymidine release. d Box and whisker graph of NEBD to anaphase duration from time-lapse movies. Statistical analysis was performed using one-way ANOVA (Kruskal–Wallis test) and Dunn's Multiple Comparison Test (for b and d data from three independent experiments n = 233 for LAP-Borealin^WT, n = 204 for LAP-Borealin^MTBM, and n = 184 for LAP-Borealin^Δ20). e Bar graph showing percent of cells undergoing anaphase with lagging chromosomes, cells were treated as described earlier in a and rescued with indicated Borealin transgene (data from n = 3 independent experiments, 63, 96, and 74 cells were analyzed for WT; 70, 84, and 50 cells were analyzed for MTBM; 68, 66, and 50 cells were analysed for Δ20). Open circle indicates the individual data points. Error bars are ±s.d. f Bar graph showing percent of cells undergoing anaphase with lagging chromosomes, treatment was done as in a, except during siRNA transfection step both Borealin and INCENP siRNA were added and the experiment was carried out in presence of 1 μg/ml Doxycycline to ensure INCENP transgene expression (data from n = 2 independent experiments, 110 and 91 cells were analyzed for INCENP^WT Borealin^WT;59 and 100 cells were analyzed for INCENP^WT Borealin^MTBM; 93 and 100 cells were analyzed for INCENP^ΔSAH Borealin^WT; 69 and 50 cells were analyzed for INCENP^ΔSAH Borealin^MTBM). Open circle indicates the individual data points. Error bars are ±s.d. Statistical analysis was performed using one-way ANOVA and Bonferroni's Multiple Comparison Test for both d and e. Bonferroni's Multiple Comparison Test was used for f. ***P < 0.001; **P < 0.01, *P < 0.05. All Box and whisker plots represent the median (central line), 25th–75th percentile (bounds of the box) and 5th–95th percentile (whiskers). Scale bar is 5 μm