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Thermal cycling over 40 cycles consisted of an initial denaturation at 94��C for 5?min, selleck screening library 94��C for 30?s, 56��C for 30?s, 72��C for 30?s, terminated by a final extension at 72��C for 5?min. GAPDH mRNA level was used for sample standardization. After electrophoresis on 1.5% agarose gel, each band was quantified by densitometer (Bio-Rad Laboratories, Hercules, CA). For phosphokinase chip array, ASCs were seeded into serum-free medium in 100?mm dishes. The following day, ASCs were incubated in hypoxia for 10?min. Cells were harvested by lysis buffer (R&D Systems) and proteins were isolated. Phosphorylation of signaling molecules was detected with a phosphokinase array kit (R&D Systems). Data are representative of triplicate independent experiments. The statistical significance of the differences among groups was tested using student's t-test. P?Angiogenesis inhibitor generated by hypoxia. The geometric mean of DCF-DA, a ROS indicator, significantly increased under hypoxia, while that of DAF-DA, a RNS indicator, was unaltered (Figure 2A, P?Quetiapine NAC treatment (Figure 2C). ROS generators such as antimycin (10?nM) and rotenone (10?pM) induced lipid accumulation (Figure 2D) and upregulated mRNA expression of PPAR-�� and C/EBP-�� expression (Figure 2E), but RNS generators such as SNAP (10?nM) and GSNO (10?nM) did not. This suggests that hypoxia increases adipocyte differentiation of ASCs mainly via ROS generation. We previously demonstrated that Nox4-produced ROS results in an increase in the proliferation and migration of ASCs. Here, we investigated whether or not Nox4 can induce an increase in adipocyte differentiation. However, a pharmacological inhibitor of Nox4 (DPI, 100?nM) or siRNA transfection of Nox4 did not reduce lipid accumulation (Supplemental Figure 1A and 1C, respectively), nor downregulation of PPAR-�� and C/EBP-�� expression (Supplemental Figure 1B and 1D, respectively), thus indicating that Nox-induced ROS generation does not mediate adipocyte differentiation.