Figure 3.
Intracellular reactive oxygen species and related genes with osteoclast differentiation treated with DFO in RAW264.7 cells. (A) Intracellular ROS levels determined using an H2DCFDA ROS probe and analyzed with FCS express flow cytometry. (B–J) Changes in the expression of the osteoclast-related genes of DFO in RAW264.7 cells. Cells were pretreated with different concentrations of DFO for 2 h followed by treatment with 50 ng/mL RANKL for 3 days. RNA was isolated from RAW264.7 cells, and cDNA was synthesized. Expression levels of tartrate-resistant acid phosphatase (TRAP), TNF receptor associated factor 6 (TRAF6), nuclear factor of activated T cell, cytoplasmic 1 (NFATc1), and cathepsin K (CTSK) were evaluated using quantitative RT-PCR relative to the RANKL treatment group (normalized to 100%). Expression levels of v-rel avian reticuloendotheliosis viral oncogene homolog A (RelA), cyclic AMP-responsive element-binding protein 1 (CREB1), peroxisome proliferator-activated receptor gamma coactivator-1beta (PGC-1β), heme oxygenase-1 (HO-1), and nuclear factor erythroid 2–related factor 2 (Nrf-2) were evaluated using quantitative RT-PCR relative to the RANKL treatment group (normalized to 100%). Data were obtained from five separate experiments, with all samples run in duplicate. The data are expressed as mean ± standard deviation values. The expression of the genes differed significantly; t-test and Mann–Whitney U test, * p < 0.05.