FIG. 1.

E. sakazakii-induced NO production is responsible for the apoptosis of IEC-6 cells. (A) Confluent monolayers of IEC-6 cells, either nontransfected or transfected with siRNA, were treated with E. sakazakii (ES) for 2 h or 4 h, the supernatants were collected and centrifuged to remove the bacteria, and NO production was determined by the Greiss method. Wild-type IEC-6 cells demonstrated a small increase in NO production by 2 h and a 10-fold increase after 6 h of exposure (*, P = 0.002), and NO production decreased significantly in siRNA-iNOS/IEC-6 cells (**, P < 0.001). The experiments were carried out separately in triplicate at least three times. (B) IEC-6 cells were infected with E. sakazakii for various periods, total RNA was extracted, and RT-PCR was performed using primers for iNOS and RPS-17. RNA expression was determined by semiquantitative PCR. In separate experiments, total cell lysates were prepared from IEC-6 cells infected with E. sakazakii for 6 h, and equal quantities of proteins were subjected to Western blotting using anti-iNOS or anti-β-actin antibody. (C) Confluent monolayers of IEC-6 cells, either nontransfected or transfected with siRNA to iNOS or eNOS, were left untreated (control) or infected with E. sakazakii for the indicated periods, and the monolayers were assessed for apoptosis by TUNEL staining using an ApoTag kit.