Effects of ATF-3 knockdown on the suppressive effects of metformin against LPS-induced MAPK phosphorylation. Control siRNA or ATF-3 siRNA was transfected into primary macrophages (A–C). Cells were then treated with metformin (M) (2 mm) for 20 h followed by LPS (L) for 30 min. Phosphorylated levels of p38 (Thr180/Tyr182) (p-p38), ERK (Tyr204) (p-ERK), JNK (Thr183/Tyr185) (p-JNK), IκBα (Ser32/36) (p-IκBα), and AMPK-α (Thr172) (p-AMPK) were determined by Western blotting. *, p < 0.05 versus control. D, ChIP-PCR was carried out after RAW264.7 cells were treated with LPS in the presence or absence of 2 mm metformin using either ATF-3 antibody or NF-κB antibody for immunoprecipitation. Relative mRNA levels of TNF-α and IL-6 were normalized versus IgG control. The experiment was repeated three times, and representative results are shown. Error bars represent S.E. *, p < 0.05 versus ATF-3 in LPS-treated cells; #, p < 0.05 versus NF-κB-p65 in LPS-treated cells. Con, control.