Figure 3

AMPK-α1-knockdown THP-1 cells exhibit impairments of TLR4-mediated signal, leading to activation of NF-κB and AP-1. (a and b) The wt THP-1 and AMPK-α1^KD THP-1 cells were transfected with or without hAMPK-α1 vector. Twenty-four hours after transfection, cells were transfected with either pBIIx-Luc (a) or AP-1-luc (b) together with Renilla luciferase vector. After 24 h, cells were treated with or without LPS (10 ng/ml) for 6 h, and then analyzed for luciferase activity. Results are expressed as the fold induction in luciferase activity relative to that of untreated cells. Error bars indicate ±S.D. of triplicate samples. (c–f) The wt THP-1 and AMPK-α1^KD THP-1 cells were transfected with or without hAMPK-α1 vector. Twenty-four hours after transfection, cells were treated with or without LPS (10 ng/ml) for 1 h, and then analyzed for DNA-binding activities for NF-KB, p65 (c) and p50 (d), and AP-1, c-Fos (e) and c-Jun (f), components as described in Materials and Methods. Results are expressed as the fold increase relative to that of wt THP-1 transfected with mock. Error bars indicate ±S.D. of triplicate samples. (g) The wt THP-1 and AMPK-α1-knockdown (AMPK-α1^KD) THP-1 cells were treated with or without LPS (10 ng/ml) for different times as indicated. The lysates were examined by western blotting with anti-pho-TAK1, anti-TAK1, anti-IκBα, anti-pho-p38, anti-p38, anti-pho-JNK, anti-JNK, anti-pho-AKT, and anti-AKT antibodies. Immunoblotting with anti-GAPDH antibody was performed to generate a control for gel loading. (h) The wt THP-1 and AMPK-α1-knockdown (AMPK-α1^KD) THP-1 cells were treated with or without LPS (10 ng/ml) for 9 h, and then analyzed for productions of TNF-α, and IL-1β, and IL-6 in supernatants using ELISA method. Error bars indicate ±S.D. of triplicate samples. *P<0.05, **P<0.01, ***P<0.001