Binding of cortactin to Arp2/3 complex and F-actin is critical for exosome secretion. (A) Cortactin domain structure indicating mutation sites: W22A, prevents Arp2/3 binding; NTA, N-terminal acidic Arp2/3-binding domain; Δ4RP, deletion of fourth repeat in tandem repeats region prevents F-actin binding; 3Y, Src kinase phosphorylation sites (tyrosines 421, 466, and 482 mutated to phenylalanine); W525K, prevents binding to SH3 domain binding partners. (B) WB analysis of cortactin wild-type (WT) and mutant (W22A, Δ4RP, 3Y, and W525K) re-expressed proteins in cortactin-KD1 cell lines. Control indicates empty vector for rescue constructs. (C) NTA quantitation of exosomes secretion rate of indicated cell lines. n ≥ 3 independent experiments. (D) NTA quantitation of exosomes secreted from SCC61 cells in the presence of DMSO or various concentrations of CK666 (Arp2/3 inhibitor) for 24 h. n ≥ 3 independent experiments. (E) Cell viability for SCC61 cells treated with DMSO or CK666 for 48 h. n ≥ 3 independent experiments. Bar graphs represent mean ± SE. *, P < 0.05; **, P < 0.01; ***, P < 0.001 indicates comparison to Sc control; ##, P < 0.01; ###, P < 0.001 indicates comparison to KD1/WT rescue. ns, not significant. Analyzed by Student’s t test.