TcdA-induced Rac1 glucosylation and cytotoxicity are dynamin-dependent.

<p><b>(A) Depletion of dynamin-1 confers resistance to TcdA challenge.</b> Caco-2 cells were transfected with 10 nM siRNA against dynamin-1 (Dyn-1) or luciferase (Luc; non-targeting control) and then intoxicated with 50 nM TcdA. ATP levels were determined using CellTiterGlo and normalized to signal from untreated cells to assess the relative survival of cells post-toxin treatment. The data represent the average of four independent experiments performed in triplicate with the SEM indicated as error bars. Data were analyzed using Welch’s t test. **p<0.005. RT-PCR confirms that siRNA treatment resulted in a decrease in Dyn-1 mRNA expression. <b>(B) Pharmacological inhibition of dynamin GTPases prevents Rac1 modification by TcdA</b>. Caco-2 monolayers were pretreated with either 80 μM dynasore or with an equal amount of DMSO control for 1 h at 37°C. Cells were switched to 4°C for 1 h and then intoxicated with either 10 nM TcdA or TcdB (positive control). Toxins were allowed to bind at 4°C for 1 h and then internalize at 37°C. TcdA and TcdB treated cells were harvested after 25 min and 15 min, respectively. Whole cell lysates were prepared for SDS PAGE and Western blot. The blot was probed with antibodies against the unglucosylated and total Rac1, and GAPDH. Cells that did not receive toxin or treatment were used as a control. <b>(C)</b> Four replicates of the experiments shown in (B) were quantified by densitometry and represented as the ratio of unglucosylated and total Rac1 levels. Results reflect the mean and SEM, and were analyzed using one-way ANOVA. p-values were generated using Dunnett’s multiple comparisons test in GraphPad Prism. **p<0.005.