TTSS-1 dependent cytotoxicity occurs later than MAPK activation. Caco-2 cells were co-incubated with viable V. parahaemolyticus WT RIMD2210633, ΔvscN1, ΔvscN2 or with heat-killed WT V. parahaemolyticus for 1, 2, 3 and 4 h (A and B) or 2 and 4 h (C and D). Values are presented as mean ± SEM; **P < 0.01 vs medium and vs WT. A: Cell lysis was determined by assaying LDH activity in the growth medium. Results are one representative experiment performed in triplicate of three independent experiments. B: MTT reduction by living cells was quantified. Results, expressed as percentage of cell viability, are one representative experiment performed in triplicate of three independent experiments. C: Cells were stained with propidium iodide to visualize dead cells with loss of membrane integrity and with Hoechst 33342 to show nuclei in all cells. Three hundred Caco-2 cells were scored via fluorescent microscopy. The results, expressed as percentage dead cells, are from three independent experiments. D: Morphological changes of the Caco-2 cells were observed by phase contrast light microscope (magnification 400×).