Cdc42 controls MTOC/IL-12 polarization at the IS. Cdc42 is recruited at the IS and is required for DC-MTCO polarization. (A) DCs were transfected with WT (Cdc42WTGFP) or dominant-negative mutant (Cdc42N17GFP) Cdc42 fused to GFP. GFP⁺ cells were enriched by cell sorting and mixed with SNARF-labeled OT-I cells (emits in the blue and red in this acquisition setting). Confocal images show a representative example of Cdc42WTGFP recruited at the IS and colocalized with polarized γ-tubulin (blue), and one example of diffused Cdc42N17GFP staining in a nonpolarized cell (Fig. S3). (B) The percentage of DCs with the MTOC polarized toward the contact region in the two groups was scored in n > 50 cells/condition in three independent experiments (**, P = 0.0124). (C) DCs were transfected with a siRNA targeted against Cdc42 (Cdc42-siRNA) or an unrelated siRNA (cont-siRNA). Protein depletion was assessed on total cell extracts by Western blot analysis at 48 and 72 h after transfection. (D) A representative confocal z slice showing the DC-MTOC position at the IS in control and Cdc42-depleted DCs (anti-γ-tubulin, red). The percentage of DCs showing a polarized MTOC was quantified on at least 90 conjugates in 3 independent experiments. Values are plotted as mean ± SEM (**, P = 0.0032 and **, P = 0.0045 at 48 and 72 h, respectively; Student’s t test). (E) Representative images and quantification of IL-12 recruitment at the IS in control and Cdc42-silenced cells. Values are plotted as mean ± SEM of two independent experiments (40 cells/condition; *, P = 0.0365, Student’s t test).