α-def-6 protects human CaCo-2 cells from intoxication with TcdA, TcdB and their combination. (A) CaCo-2 cells were treated with TcdA (500 pM), TcdB (50 pM) or the combination of both toxins in the presence and absence of α-def-6 (6 µM). For control, the cells were left untreated. Representative images after 6 h of incubation time are shown. Scale bars correspond to 100 µm. (B) CaCo-2 cells were treated as in (A). Images were taken each hour, and the amount of rounded cells over time was determined. Values are given as mean ± SD of three technical replicates. Three biological replicates showed comparable intoxication and inhibition (n = 3). (C) Transepithelial electrical resistance (TEER) was measured across a CaCo-2 monolayer. Cells were seeded in cell culture inserts (for TcdB: Millicell; for TcdA and TcdA + TcdB: Brand), and the toxins and α-def-6 (6 µM) were added apically. For TcdB (100 pM), a time-dependent protection of the CaCo-2 monolayer in the presence of α-def-6 is displayed (left panel). For TcdA (200 pM) and TcdA + TcdB, data after 2 h are shown (right panel). Values are given as mean ± SD. Significance was determined using a one-way ANOVA combined with a Dunnett’s multiple comparison test (** p < 0.01, *** p < 0.001). Three biological replicates showed comparable results (n = 3).
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