FIG 2.

Rab5a shRNA inhibits CdtB-induced cytotoxicity and apoptosis in human colonic epithelial cells. Stable primary human colonic epithelial cells, with Rab5a shRNA (“S1/S2,” two different sequences) or the scrambled shRNA (sc-shRNA), were either left untreated (“Ctrl,” same for all figures) or treated with Cdt holotoxin (2 μg/ml) for the applied time, and the expression of the listed genes was tested by qPCR (A) and Western blotting (B); cell viability and cell death were tested by an MTT assay (C) and an LDH medium release assay (D), respectively. Caspase-3/-9 activation (E and F) and cell apoptosis (G) were tested by the assays mentioned in the text. Rab5a mRNA was normalized to GAPDH mRNA (same for all figures). Rab5a and cellugyrin protein expression were normalized to the loading control GAPDH/tubulin (B and F). Cell apoptosis was tested by the annexin V-FACS assay (H and I). Bars represent means ± SD (same for all figures). *, P < 0.05 versus Ctrl treatment of sc-shRNA cells. ^#, P < 0.05 versus CdtB treatment of sc-shRNA cells. The experiments reflected in this figure were repeated three times, and similar results were obtained.