Figure 4
PTL and MCL inhibit the NF-κB pathway. (a) RAW 264.7 cells were transfected with the luciferase reporter construct, pNF-κB-RE-LUC, and treated with LPS (100 ng/ml) in combination with 0, 1, 2, or 5 μM PTL or MCL. Cells were lysed and luciferase activity was assessed. *P<0.01 compared with cells stimulated with LPS-only. (b) RAW 264.7 cells were treated with 5 μM PTL or MCL for 8 h and then stimulated for 0, 10, 20, 40, or 60 min with 10 μg/ml of LPS. Activation of the NF-κB pathway was analyzed by quantifying total and phosphorylated NF-κB and total and phosphorylated IκBα proteins using western blotting. Bar graph represents densitometric quantifications of phosphorylated-NFκBα western blot normalized to β-actin. Values represent means±s.e.m. of three blots from independent experiments. (c) The translocation of NF-κB-p65 to the nucleus was assessed by immunofluorescence. RAW 264.7 were incubated for 8 h with 5 μM of PTL, MCL, or the DMSO control and then induced by 10 μg/ml of LPS for 30 min. RAW 264.7 were immunostained using anti-NF-κB-p65 and cell nuclei were stained using DAPI, scale bars=20 μm. (d), The percentage of NF-κB-p65 translocation to the nuclei was determined.
