Degraded Carrageenan Causing Colitis in Rats Induces TNF Secretion and ICAM-1 Upregulation in Monocytes through NF-κB Activation
Figure 7
Degraded CGN activated the NF-kB pathway in monocytes.
A: THP-1 cells were transfected with a NF-κB reporter plasmid driving expression of luciferase. Cells were then treated with various concentrations of 10 kDa (triangles), or 40 kDa dCGN (squares). B: THP-1 cells treated with 1 mg/ml of 10 kDa dCGN (C10), or with 1 mg/ml of 40 kDa dCGN (C40) were lysed after various periods of time. Proteins in cell extracts were resolved by SDS-PAGE and then Western blotted for IκBα or α−tubulin as loading control. C: Degraded carrageenans (dCGN) induced activation of NF-κB. THP-1 cells were treated with nothing (control), or with 1 mg/ml of 10 kDa dCGN (C10), or with 1 mg/ml of 40 kDa dCGN (C40) for 30 minutes at 37°C. Nuclei were isolated and lysed. Proteins in nuclear extracts were resolved by SDS-PAGE and then Western blotted for NF-κB p50 subunit (p50) or NF-κB p65 subunit (p65). Lower panels show Western blots of nuclear ERK revealing equivalent amount of protein in each sample. Data are representative of three separate experiments. D: Degraded carrageenan (dCGN) induced activation of NF-κB. Nuclei isolated from THP-1 cells were fluorescence-stained for NF-κB p50 subunit or NF-κB p65 subunit before (filled area) or after cells were treated with 1 mg/ml of 10 kDa dCGN (C10), or with 1 mg/ml of 40 kDa dCGN (C40) for 30 minutes at 37°C. Dashed line corresponds to nuclei stained only with secondary fluorescence antibody. Fluorescence intensity was analyzed by flow cytometry as described.