Fig. 2: Requirement of CD99 for microtubule reorganization, and the physical interaction between actin and microtubules.

a Confocal images of WT- and Cd99^−/−-cells stained with anti-α-tubulin antibody at 15 min after anti-CD3-stimulation. Plots showing the number of perpendicularly growing microtubules (pMT) in the cell body (CB) and lamellipodia (LP) (n = 12 cells per group), and cellular microtubule (MT) fluorescence intensity (n = 24 cells per group). b, c Longitudinal analysis of microtubule reorganization in Lifeact⁺ WT- and Cd99^−/−-cells (see Supplementary Movies 2, 3). Time-lapse images of microtubules overlaid on DIC (b) and F-actin (c) images. Actin-microtubule colocalization shown in white. Time in minutes:seconds. Plots of microtubule staining (SiR-tubulin⁺) intensities and pMT numbers (n = 5 cells per group) (b), and actin-microtubule colocalization according to Manders’ coefficient of microtubules colocalized with F-actin (n = 3 cells per group) (c). d Confocal images of F-actin (red) and microtubules (green), and their colocalization (yellow) in WT- and Cd99^−/−-cells stained with phalloidin and anti-α-tubulin antibody at 15 min after CD3-stimulation. Plot of actin-microtubule colocalization (n = 12 cells per group). e Immunoblot of actin in coprecipitates of α-tubulin from stimulated WT- or Cd99^−/−-cells. Data a–e are representative of at least three independent experiments. Data a–d are shown as mean ± SEM. Statistical significance was assessed using unpaired two-tailed t-test (a, b, d), and two-way repeated measures ANOVA with Sidak post-hoc test (b, c). Exact p-values are presented in the figure. Source data are provided as a Source Data file.