The mDia2 protein is required for loading new CENP-A during G1. (A) High-resolution ratiometric live-cell imaging showing defective YFP-CENP-A loading upon mDia2 knockdown. Pseudocolored live imaging stills following cells through the 10-h time window after anaphase onset. Identical lookup table (LUT, linear and covering the full range of data) was used over time. Bar, 10 µm (insets are 3× magnified). (B) Quantification of centromeric YFP-CENP-A levels during G1 phase (plotted as mean ± SD). Control: n = 4,100 centromeres from 12 G1 pairs, and mDia2 siRNA: n = 4,310 centromeres from 13 G1-pairs were measured from three independent transfections (see Materials and methods and Fig. S2 for more details). (C) Scheme for the SNAP pulse chase labeling to distinguish existing centromeric CENP-A protein (old) from newly synthesized CENP-A loaded onto centromeres (new). (D) Immunofluorescence analysis showing old CENP-A labeled by TMR-Star and total CENP-A stained with anti-HA antibody. In merge: red, TMR-Star; green, HA-tag. Bars: (main) 10 µm; (insets) 1 µm. (E) Quantification of SNAP-tag-labeled CENP-A (old, red) and total CENP-A (total, green; means ± 95% confidence intervals). The p-value was computed using a two-tailed t test. (F) Quantification showing the normalized ratio between total CENP-A and old CENP-A (mean ± SD overlaid with scatterplot). Control: n = 215, and mDia2 siRNA: n = 217 cells from three independent experiments. The p-value was computed using a two-tailed t test.