Figure 7. Inhibition of IbeA+ E. coli K1-induced pathogenicities, phosphorylation of ERK/IKK and nuclear translocation of NF-κB by knockdown of PSF.

HBMECs were transfected with PSF or control siRNA as described in Materials and Methods. IbeA+ E. coli K1 penetration (A) and PMN transmigration (B) across siRNA-transfected HBMECs were performed as described in the Materials and Methods. Both invasion and PMN transmigration assays were performed in triplicates. Results for invasion are expressed as a relative percentage compared to the penetration rate of E44 in the siRNA control (CON) (100%). Results for PMN transmigration are expressed as the percentage of PMN transmigration of total PMNs. The control siRNA-transfected HBMECs infected with E44 and ZD1 were used as the controls (panels A and B). The significant differences regarding to the control were marked by asterisks (*P<0.05; **P<0.01). (C) After transfection, the cells were stimulated with E44 or ZD1 (10⁷/ml) for 30 min or 2 h. PSF re-localization, p-Erk1/2, p-IKK α/β and IκBα degradation were examined in cytoplasmic fractions after 30 min of stimulation with E. coli K1 strains. NF-κB (p65) and PSF in nuclear fractions were examined after 2 h of incubation with E. coli K1 strains. β-actin in both fractions was detected as internal loading controls. Control: HBMECs transfected with control siRNA; PSF KD, HBMECs transfected with PSF siRNA; UNT: Untreated HBMECs. (D) Time course analysis of IbeA-induced tyrosine phosphorylation of PSF. HBMECs were incubated with the IbeA protein (0.1 µg/ml) for 2, 6, and 24 hrs, respectively. The cytoplasmic fractions were extracted and immunoprecipitated (IP) using the anti-phosphotyrosine antibody as described in Materials and Methods. The Tyr-IP complexes were subjected to Western blot using the mouse monoclonal antibody against PSF. Total mouse IgG was detected as an internal loading control. CON: the IP control without primary antibody incubation; 0 h: the control HBMECs without IbeA incubation.