Figure 5.

Analysis of the interaction between SIC and THP1 cells

(A) THP1 cells were incubated for 45 min with 5 μg/mL AlexaFluor 633-labeled SIC protein (red). Live imaging of the cells was used to observe the interaction of SIC with the THP1 cells. Scale bar represents 20 μm.

(B–E) Flow cytometry analysis of SIC-monocyte interaction. B: THP1 cells incubated with medium (Ctr) C: THP1 cells incubated with 5 μg/mL SIC (SIC) D: THP1 cells incubated with 5 μg/mL SIC on ice (SIC ice) E: THP1 cells pre-incubated with 20 μM CytD for 30 min, then incubated with 5 μg/mL SIC (SIC CtdD).

(F) Median fluorescent intensity analysis of SIC interaction with the cells in B-E. Data shown from 3 independent experiments, ∗p < 0.05, ∗∗p < 0.01.

(G) THP1 cells, untreated or trypsinized, were incubated with 5 μg/mL SIC +/− 2.5% plasma (P). Lysates of the cells were subjected to SDS-PAGE analysis and Western blot. Membranes were probed with antibodies against p38 MAPK (p38) and phosphorylated p38 MAPK (p-p38). Band intensities were analyzed.

(H) TNFα secretion by untreated and trypsinized THP1 cells, incubated with 5 μg/mL by SIC +/− 2.5% plasma (P) was measured by ELISA at 450 nm.

(I) Untreated and trypsinized THP1 cells were incubated with 5 μg/mL SIC +/− 2.5% plasma (P) and activation of NF-κB was measured at 655 nm.

(J) Untreated and trypsinized THP1 cells were incubated with 5 μg/mL SIC fragment II +/− 2.5% plasma (P) and activation of NF-κB was measured. All data represent mean ± SEM of 3-5 independent experiments, one-way ANOVA, Dunnett's multiple comparison test, with single pooled variance ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001.