Figure 3: PEDF expression with up-regulation of NADPH oxidase. The adenovirus expressing the constitutively active form of PU.1 transcription factor was transfected into 3T3-L1 adipocytes and levels of PU.1 protein expression were measured (A, upper panel), as well as transcription levels of two isoforms of NADPH oxidase, p22 and p47 (A, lower panel). After stimulating 3T3-L1 adipocytes with 1 mM FFA for 12 hr, these cells were washed twice in PBS, followed by addition of 10 μM CM-H2DCFDA in PBS, placed in the dark at 37°C for 1 hr, then harvested and suspended in 500 μl of PBS. ROS levels were measured flow cytometrically by determining the mean fluorescent intensity relative to that of the control group (B, upper panel). Corresponding to the cells used for the ROS assay, PEDF expression was examined by Q-PCR (B, lower panel). Representative data from four independent experiments are presented. *Significant difference (P <0.05) relative to control cells. N.S.; not significant relative to control cells.