Fig. 2.

MITD1 has a role in cytokinesis. (A) HeLa cells were transfected with two different siRNAs against MITD1 or a luciferase control and scored for multinucleation or visible midbodies. Depletion of hIST1 was used as a positive control. Depletion of MITD1 was verified by Western blot using anti-MITD1 antibody. Anti-HSP90 was used as a loading control. Error bars indicate the SD from the mean for three independent measurements. (B–D) Asynchronous cultures of HeLa cells stably expressing mCherry-Tubulin were transfected with the indicated siRNA and imaged live. (B) Quantification of the time before a dividing cell coalesces into a multinucleated cell. The red line represents the median time. (C) The median abscission time for cells that divided without becoming multinucleated was calculated across six independent experiments [Non Targeting Control (NT CTRL): 80 ± 44.4 min, n = 667; MITD1-1: 70 ± 56.9 min, n = 906; hIST1: 160 ± 111.2 min, n = 357]. The differences in abscission times are significant (P < 0.0001) as determined by a Mann–Whitney u test. (D) Quantification of the percentage of cells that displayed pronounced plasma membrane blebbing. (E and F) Confocal fluorescence images of HeLa cells stably expressing HA-MITD1 alone (E) or in combination with YFP-Tubulin (F). Cells were doubly stained with anti-HA and either anti-tubulin (E), anti-hIST1 (F, Upper) or anti-MKLP1 (F, Lower) antibodies. In the overlays, DNA is shown in blue, MITD1 in green, and tubulin in red (E) or MITD1 in red and hIST1/MKLP1 in blue (F). In all cases, the inset shows a magnification of the midbody.