Fig. 2.

N,N-dimethylformamide (DMF) inhibits high glucose-induced reactive oxygen species (ROS) accumulation. a) The glucose-treated (5 mM or 40 mM) bone marrow macrophages (BMMs) were preloaded with dichloro-dihydro-fluorescein diacetate (DCFH-DA) and treated with 50 ng/ml receptor activator of nuclear factor κB ligand (RANKL), 20 μM DMF, or their combination (×400). The ROS levels were assessed by flow cytometry analysis. The representative microscopic fields are shown: b) the relative intensity of DCF fluorescence in BMMs and d) RAW 264.7 cells. The c) glucose-treated (5 mM or 40 mM) BMMs and e) RAW 264.7 cells were treated with 50 ng/ml RANKL, 20 μM DMF, or a combination of the two. ROS levels were assessed by flow cytometry analysis. The glucose-treated (5 mM or 40 mM) RAW 264.7 cells were treated with DMF at the indicated dose. f) The messenger RNA (mRNA) levels of Gclc, Gclm, Ho-1, and Nqo1 were examined by quantitative polymerase chain reaction. g) The protein levels of GCS, HO-1, and NQO1 were tested by Western blot. h) Western blot results were quantified by ImageJ software (National Institutes of Health, USA). Data were presented as mean and standard deviation of three independent experiments. *p < 0.05 versus control of the same glucose concentration group, #p < 0.05 versus 5 mM glucose with the same DMF and/or RANKL treatment. $p < 0.05 versus the same glucose concentration plus RANKL treatment (two-way analysis of variance).