Fig. 3: The activity of Rho is critical for migrasome formation.

a The phylogenetic tree of mouse small GTPases. Amino acid sequences of 25 mouse small GTPases were aligned, and the phylogenetic tree was generated by MEGA software based on the Neighbor-Joining algorithm. Small GTPases that are targeted by specific toxins are labeled with colored boxes. The scale bar represents units of evolutionary distance. b Quantification of the numbers of migrasomes per field from multiple small GTPases knock-down cell lines. Data are presented as mean ± SEM; n = 24–28. P values were calculated using ordinary one-way ANOVA followed by Dunnett’s test. c Confocal images of NRK cells stably expressing TSPAN4-mCherry treated with cytoskeleton inhibitors and TcdB3 together. Cells were pretreated with Latrunculin A, nocodazole, and taxol for 30 min, and subsequently treated with 0.2 pM TcdB3 and inhibitors together for 3–4 h. Red circles indicate actual migrasomes. Scale bar, 20 μm. d Quantification of the numbers of migrasome from c. Data are presented as mean ± SEM; n = 25–29. P values were calculated using ordinary one-way ANOVA followed by Dunnett’s test.