(A) Streptavidin-latex beads were coated with biotinylated mycolactone or solvent control, then incubated with N-WASP and placed in Jurkat T cell extracts supplemented with ATP, MgCl₂, and Alexa Fluor 488–labeled actin monomers for 2 hours. Representative beads are shown in phase-contrast and fluorescence. Mean fluorescence signal of more than 30 beads from 3 independent experiments are compared. ****P < 0.0001, unpaired 2-tailed t test, Welch corrected. (B) Jurkat cells were treated with a mycolactone fluorescent derivative (bodipy-Myco) for 1 hour, then processed for immunofluorescence with antibody binding the open form of WASP (active-WASP) and antibody recognizing p34-ARP2/3. Each image corresponds to a single confocal plane. 8 of 11 randomly picked cells had a Pearson coefficient greater than 0.5 (calculated on a z stack), indicative of Bodipy-mycolactone and active WASP colocalization. (C) Differential ARP2/3 complex recruitment in HeLa cells treated with vehicle (control), 20 nM mycolactone for 4 hours, or mycolactone plus 1 μM wiskostatin (Wisko). Representative immunofluorescence images and integrated p34-ARP2/3 intensities (n > 50) in the perinuclear region (see Methods) are shown. Perinuclear enrichment was calculated as mean ± SEM intensity and presented relative to control. *P < 0.05, ANOVA with Dunn post-test. (D) Western blot analysis of N-WASP, p34-ARP2/3, and actin expression in HeLa cells exposed to 20 nM mycolactone for 6 or 16 hours, compared with vehicle-treated control cells and untreated cells (Unt). GAPDH served as an internal control. Scale bars: 5 μm (A and B); 25 μm (C).