Figure 3: LITAF promotes Extracellular Vesicle production and α-toxin trafficking to multivesicular bodies.

(A-B) Quantification of exosomes secreted by wild type and LITAF-expressing U2OS cells with and without treatment with α-toxin (500 ng/ml, 2 hrs). (A) Representative histogram of CD9 fluorescence. (B) Exosome production expressed as percentage of α-toxin-treated wild-type U2OS cells. (n = 5 replicate / condition), combined from five independent experiments. **, p<0.01; one-way ANOVA. (C) Representative immunoblot of exosome fractions stained for α-toxin, Tsg101, Alix, LITAF and EEA1. Bottom panel shows immunoblot of equivalent cell lysates stained for EEA1 and actin. (D) Representative confocal images of GFP-LITAF (green) and nucleus (white) in U2OS cells, untreated or following α-toxin treatment (500 ng/ml, 1h). (E) Relative numbers of U2OS cells untreated or α-toxin treated as in (D) with LITAF localized predominantly to the plasma membrane or predominantly in internal vesicles from analysis of ≥ 100 cells per condition. Mean ± SD of n≥3 replicate wells/condition, representative of three independent experiments (F) Representative confocal images of U2OS cells expressing GFP-LITAF and RFP-Rab5, untreated or treated with α-toxin (500 ng/ml, 1h) and stained for CD63. Regions within dotted boxes are enlarged in adjacent images. (G) Pixel intensity of indicated stains along the dotted line in (F) (H) Co-localization (Pearson’s correlation) of LITAF and Rab5, or LITAF and CD63, in GFP-LITAF-expressing U2OS cells untreated or treated with α-toxin (500 ng/ml, 1h). Each point represents a single image of n≥6 replicate images/condition, combined from four independent experiments. (I) Representative confocal images of U2OS cells treated with α-toxin (500 ng/ml, 1h) and stained for α-toxin, CD63 and LBPA. Regions within dotted boxes are enlarged in adjacent images. (J) Pixel intensity of indicated stains along the dotted lines in enlarged panels in (I). (K) Co-localization (Pearson’s correlation) of α-toxin and CD63, or α-toxin and LBPA in control and LITAF-expressing U2OS cells following treatment with α-toxin as in (E). Each point represents a single image of n≥6 replicate images/condition, representative of three independent experiments. (L) Representative confocal images of α-toxin-treated U2OS cells stained for LITAF and α-toxin. Region in dotted box is enlarged in adjacent images (M) Pixel intensity of indicated stains along the dotted line in (L) Scale bar in all images is 10 μm. * p < 0.05, ** p < 0.01, **** p <0.0001, Unpaired Student’s t test (H,K) or two-way ANOVA (E).