Figure 4: Uptake of exogenous CnB did not occur via binding to LPS, and CnB induced cytokine secretion.

(a) The uptake of CnB could be inhibited by LPS. (b) The uptake of LPS could be inhibited by CnB. A 30-fold excess of LPS or CnB was mixed with CnB-GFP or rhodamine-labelled LPS and incubated with the SK-HEP-1 cells for 30 min. The results were visualized using an inverted fluorescence microscope (upper panel, scale bar 50 μm, 20×) and quantified using a microplate reader (lower panel). (c) CnB-GFP did not bind to LPS. To a black ELISA plate, 10 μg/ml LPS was immobilized and incubated with different concentrations of CnB-GFP, DyLight 488-labeled CD14 or GFP to evaluate the binding between CnB and LPS. (d) The CnB-induced cytokine production was not due to LPS contamination. The RAW264.7 cells were incubated with 1 μg/ml LPS, 100 μg/ml CnB, 1 μg/ml LPS in the presence of 100 μg/ml polymyxin B or 100 μg/ml CnB in the presence of 100 μg/ml polymyxin B for 24 h and the levels of the secreted cytokines in the supernatant were measured by ELISA. (e) NF-κB was activated by CnB in the TLR4-transfected Hek293 cells. The Hek293 cells were transfected with the TLR4-pcDNA3.1 plasmid and incubated for 24 h. The cells were plated in 24-well plates (1 × 10⁵/well) and co-transfected with the pNF-κB-luc and pRL-null-Renilla-luc plasmids. Twenty-four hours post-transfection, the cells were co-incubated with 1 μg/ml LPS, 400 μg/ml CnB, 1 μg/ml LPS in the presence of 100 μg/ml polymyxin B, 400 μg/ml CnB in the presence of 100 μg/ml polymyxin B, 1 μg/ml proteinase K-treated LPS or 400 μg/ml proteinase K-treated CnB for 12 h. Luciferase activity was measured using a Dual-luciferase Reporter system. The data were normalized to the control. Data represent three independent experiments (mean ± s.e.m., n = 3). *P < 0.05, **P < 0.01, ***P < 0.005 (t-test, two-tailed).