Cdc42 localizes near centrosomes during mitosis. The localization of Cdc42 in mitosis depends on ITSN2. (A) Confocal images of Cdc42 localization in MDCK cells in different phases of the cell cycle. MDCK Cdc42-GFP cells were plated in a monolayer and stained for acetylated tubulin (Ace Tub) and chromatin (blue). Cells in interphase (top), metaphase (middle), and anaphase (bottom) are shown. (B) Cdc42 levels are regulated in Cdc42-GFP cells. Total cell lysates of wild-type MDCK cells and Cdc42-GFP cells were immunoblotted for Cdc42, and total Cdc42 levels were quantified using α-tubulin as a control. Molecular mass is indicated in kilodaltons. Values shown are mean ± SD from three different experiments (Western blots). (C) Confocal images of ITSN2 colocalizing with Cdc42 in dividing cells. Cdc42-GFP cells were transfected with ITSN2-Cherry (red) and stained for α-tubulin (α-tub). Arrows indicate the localization of ITSN2 and Cdc42. (D) Confocal images showing that the localization of Cdc42 in mitosis depends on ITSN2. MDCK cells stably expressing Cdc42-GFP were silenced with siRNA oligonucleotides to ITSN2 (right) or control (left) and plated to form cysts for 24 h. Cells with Cdc42-GFP were stained for acetylated tubulin and chromatin (blue). (A and D) Arrows indicate Cdc42 localization. (E) Quantification of Cdc42-GFP associated with the mitotic spindles in control cell or cells silenced for ITSN2. Cells with Cdc42-GFP concentrated at the spindle poles or dispersed throughout the cytoplasm were quantified. Values shown are mean ± SD from three different experiments (n = 100 cysts/experiment; *, P < 0.001 for cytoplasmic/pericentrosomal Cdc42-GFP localization in ITSN2 KD vs. control cells). Bars, 5 µm.