Fig. 1: C. difficile TcdB3 induces migrasome formation in cultured cells.

a Confocal image of the WT NRK cells staining by WGA treated with 0.2 pM TcdB3 or not for 4 h. Scale bar, 10 μm. b A representative SEM image of NRK cells treated with 0.2 pM TcdB3 for 4 h. An enlarged ROI was shown in the up-left corner. Scale bar, 500 nm. c Representative TEM images of NRK cells treated with 0.2 pM TcdB3 for 4 h. Scale bar, 500 nm. d Confocal images of the WT, TSPAN4 knockout, and TSPAN4-mCherry overexpressing NRK cells, which were treated with 0.2 pM TcdB3 for 4 h. The WT and TSPAN4 knockout cells were stained by WGA-AF488. Scale bar, 20 μm. e Quantification of migrasomes per field from d. Data are presented as mean ± SEM; n = 18. P values were calculated using ordinary one-way ANOVA followed by Dunnett’s test. f Time-lapse confocal imaging of NRK cells stably expressing TSPAN4-GFP treated with 0.2 pM TcdB3. The gray bar indicates the time scale of 630 min and the specific time points of each picture were labeled and marked as a short slash inside the gray time scale. Scale bar, 10 μm.