Stx2-positive microvesicles are toxic to Gb3-positive but not Gb3-negative cells. CHO-Gb3, CHO-control and CHO native cells, HeLa and HeLa-PPMP and DLD-1 cells were incubated with Shiga toxin 2 positive microvesicles (Stx2-pos MVs), Stx2-negative microvesicles (Stx2-neg MVs), Stx2 or PBS and protein synthesis was measured. (A) CHO-Gb3 cells were incubated with Stx2-positive microvesicles (n = 12), Stx2-negative microvesicles (n = 12), free Stx2 (n = 14), or PBS (n = 18). A significant decrease in protein synthesis was seen in both Stx2-positive microvesicles and free Stx2 compared to Stx2-negative microvesicles (2 independent experiments). (B) CHO-control cells were treated with Stx2-positive microvesicles (n = 12), Stx2-negative microvesicles (n = 12), free Stx2 (n = 6), or PBS (n = 10). No differences in protein synthesis were noted between the groups (one experiment). (C) CHO native cells were incubated with Stx2-positive microvesicles (n = 18), Stx2-negative microvesicles (n = 18), free Stx2 (n = 12), or PBS (n = 13). No differences in protein synthesis were noted between the groups (three independent experiments). (D) HeLa cells were incubated with Stx2-positive microvesicles (n = 12), Stx2-negative microvesicles (n = 12), free Stx2 (n = 16), or PBS (n = 18). Cells treated with Stx2-positive microvesicles and free Stx2 exhibited significantly lower protein synthesis compared to untreated and Stx2-negative microvesicle-treated cells (two independent experiments). (E) HeLa-PPMP cells were treated with Stx2-positive microvesicles (n = 6), Stx2-negative microvesicles (n = 6), free Stx2 (n = 6), or PBS (n = 6). No difference in protein synthesis was seen between the groups (one experiment). (F) DLD-1 cells were incubated with Stx2-positive microvesicles (n = 5), Stx2-negative microvesicles (n = 5), free Stx2 (n = 9), or PBS (n = 8). No differences in protein synthesis were seen between the groups (two independent experiments). Median protein synthesis per cell is denoted by the bar. The median protein synthesis of untreated cells was defined as 100% in each experiment. *P < 0.05, **P < 0.01, ****P < 0.0001, Kruskal-Wallis test.