The formin mDia2 is a downstream effector of the MgcRacGAP-dependent GTPase pathway to regulate epigenetic centromere maintenance. (A) HeLa cells 48 h after transfection of indicated siRNAs along with expression vectors were fixed and stained with DAPI (DNA), CENP-B, and CENP-A. Transfected cells were identified by fluorescence markers. Bar, 10 µm. (B and D) Quantifications of normalized CENP-A integrated intensity per nucleus plotted as means ± SD overlaid with scatterplot. The p-value was computed using a two-tailed t test. Control siRNA: n = 425, mDia2 siRNA: n = 260, mDia2 siRNA + FL-mDia2: n = 136, mDia2 siRNA + WT-FH1FH2-mDia2: n = 383, mDia2 siRNA + K853A-FH1FH2-mDia2: n = 151, mDia2 siRNA + I704A-FH1FH2-mDia2: n = 158, and mDia2 siRNA + W630A-FH1FH2-mDia2: n = 128. Control shRNA: n = 135, MgcRacGAP shRNA: n = 117, and MgcRacGAP shRNA + WT-FH1FH2-mDia2: n = 88 from at least three independent experiments. (C) Live-cell imaging stills showing EGFP-FH1FH2-mDia2 nuclear localization during G1 phase upon anaphase onset (0 min). Bar, 10 µm.