Figure 1: IL-27 treatment enhances NADPH oxidase-2-mediated superoxide production in macrophages.
(a–d) Superoxide Production was monitored by detecting H2O2 amounts after stimulation with PMA as described in the Method section. (a) M-Mac were treated with 0 or 100 ng/ml of IL-27 for 24, 48 or 72 h followed by stimulation with PMA, (b) M-Mac were treated with 100 ng/ml of IL-27 for 48 h and then stimulated with PMA for 5, 10, 20, or 30 min at 37 °C. (c,d) M-Mac was cultured with 100 ng/ml IL-27 for 48 h, and then pretreated with apocynin or DPI for 30 min before PMA stimulation (e,f) M-Mac were treated with 100 ng/ml of IL-27 for 48 h. (e) the expression of each subunit mRNA of NADPH oxidase-2 was determined by quantitative RT-PCR. Data shown means ± SE of three independent studies. (f) The expression of p47phox protein was detected by Western blot. (g) M-Mac were treated with 0 or 100 ng/ml of IL-27 for different durations, or (h) different doses of IL-27 for 48 h. The relative level of expression of p47phox gene was measured by quantitative RT-PCR. (i,j) M-Mac were treated with CHX for 30 min at 37 °C and then cultured in the presence of 0 or 100 ng/ml of IL-27 for 48 h. The expression of p47phox protein (i) and mRNA (j) were analyzed by Western blot and quantitative RT-PCR, respectively. (k) M-Mac were incubated with 0 or 100 ng/ml of IL-27 for 48 h and then SOD isotype expression was analyzed by Western blot. (l) M-Mac were treated with 0 or 100 ng/ml of IL-27 for 48 h and then infected with HIV. The HIV infected cells were cultured for 14 days and then HIV replication was monitored by p24 antigen capture kit. Data shown represent means ± SDs of triplicate samples of two independent experiments. *P < 0.01, **P < 0.05 In Western blot analysis, anti-b-actin antibody was used to determine an internal control.
