Depletion of MISP impairs the metaphase-to-anaphase transition. (A) Asynchronous HeLa cells were stained for p-Ser10 H3 (red) and α-tubulin (green) 48 h after control or MISP Ol2 siRNA treatment. Bar, 50 µm. (B) Quantification of the mitotic index in HeLa cells transfected with control or MISP siRNA (Ol1/Ol2) by evaluating p-Ser10 H3 staining. Results represent the mean of three different experiments (n = 200 cells per experiment). (C) HeLa cells stably expressing GFP-α-tubulin/RFP-H2B were transfected with control or MISP Ol2 for 24 h followed by live-cell imaging for 8 h. Frame series of movies of control and MISP knockdown cells with continuous time points (minute) are shown. Bar, 10 µm. (D) Box plot of C displaying the time from NEB to anaphase; n = 20 cells each from three independent experiments. (E) Percentage of mitotic cells with aligned chromosomes during early mitosis in control and MISP siRNA-treated cells evaluated by live-cell imaging (n = 100 cells from two independent experiments). (F) HeLa cells were treated with control or MISP siRNAs with and without additional depletion of Mad2 for 48 h. Samples were analyzed by Western blot. Arrowhead indicates phosphorylated BubR1 with retarded electrophoretic mobility. (G) HeLa cells transiently transfected with GFP alone or siRNA-resistant versions of GFP-MISP WT/7AP/9AC/6DP were treated with MISP or control siRNA for 48 h. The mitotic index was evaluated (n = 200 cells) by p-Ser10 H3 staining, respectively from three independent experiments. Error bars in D, E, and G represent SD. Student’s t test was used to calculate p-value for comparison of control- and MISP-depleted samples.