Figure 3

HMGB1-activated AKT/GSK3β/β-catenin signaling pathways.

(a) BEAS-2B cells were treated with different doses of HMGB1 for 10 min and expression of different proteins was detected by western blot. (b) BEAS-2B cells were treated with HMGB1 (300 ng/mL) for different periods and analyzed for expression of different proteins by western blot. (c) BEAS-2B cells were treated with HMGB1 (300 ng/mL) for different periods and analyzed for β-catenin expression by western blot. (d) BEAS-2B cells were treated with HMGB1 (300 ng/mL) for different periods and analyzed for β-catenin mRNA expression by real-time quantitative PCR. (e) BEAS-2B cells were treated with HMGB1 (300 ng/mL) for different periods and analyzed for nuclear and cytosolic β-catenin expression by western blot. (f) BEAS-2B cells were treated with 10 μM PI3K inhibitor (LY294002) and GSK-3β inhibitor (SB415286) for 10 min and then treated with HMGB1 (300 ng/mL) for 24 h. β-catenin expression was assessed by western blot analysis. Quantification of protein expression was performed using ImageJ software. Data are expressed as mean ± SD. *p < 0.05, **p < 0.01, as compared with the control group. #p < 0.05, as compared the HMGB1-treatment group. (A,B) (n = 4); (C–F) (n = 3). (g) Immunofluorescence staining of β-catenin (green) was detected by fluorescence microscopy. Data are expressed as mean ± SD (n = 4). *p < 0.05, as compared with the 0-min group. In immunoblotting assay, gels have been run under the same experimental conditions. Then cropped blots were incubated with different primary antibodies for analysis of signaling pathway.