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. 2008 Dec 12;283(50):34833–34843. doi: 10.1074/jbc.M805866200

FIGURE 4.

FIGURE 4.

eNampt induces STAT3 phosphorylation by promoting the secretion of an autocrine/paracrine STAT3 activator, interleukin-6. A, left blot, macrophages were incubated in the absence (Con) or presence of 100 ng/ml eNampt (eNmt) for 24 h, and cell lysates were immunoblotted for tyrosine-phosphorylated and total STAT3. Right blot, for these experiments, CM from control or eNampt-treated macrophages was transferred to fresh macrophages. The cells were then incubated in this medium for the indicated periods of time, after which cell lysates were immunoblotted for tyrosine-phosphorylated and total STAT3. B, macrophages were incubated in the absence or presence of 100 ng/ml eNampt for 5 h. The cells were washed and then incubated in fresh medium for 12 h. This “chase” conditioned medium (CM) was then transferred onto fresh macrophages. After 30 min of incubation, cell lysates were immunoblotted for tyrosine-phosphorylated STAT3 (pY-STAT3) and β-actin. C, macrophages were incubated in the absence or presence of 100 ng/ml eNampt for the indicated times. The culture media were collected and cleared of cell debris by centrifugation, and concentrations of IL-6 and IL-10 were measured by an enzyme-linked immunosorbent assay. D, eNampt-conditioned media were prepared as in A and then transferred to recipient macrophages that had been pretreated for 30 min with 1 μg/ml anti-IL-10 receptor IgG, anti-IL-6 receptor IgG, or nonimmune IgG control. The recipient cells were incubated for 30 min with the conditioned medium plus the respective IgGs. Cells were lysed, and the extracts were immunoblotted for tyrosine-phosphorylated and total STAT3. E, eNampt or control “chase” CM were prepared as in B. The CM was incubated with or without 1 μg/ml anti-IL-6 antibody for 30 min. The media were then added to recipient macrophages and incubated for 30 min. 100 pg/ml recombinant IL-6 and IL-6 plus anti-IL-6 antibody were used as positive and negative controls, respectively. Cells were lysed, and the extracts were immunoblotted for tyrosine-phosphorylated STAT3 and β-actin. F, in both experiments shown, cholesterol-induced apoptosis was assayed in control or eNampt-pretreated macrophages exactly as in Fig. 1A, except some of the cells were incubated with 1 μg/ml anti-IL-6 antibody, anti-IL-10 antibody, or both throughout both the 24-h eNampt pretreatment period and the 20-h cholesterol-loading period. In experiment 1, p < 0.01 for cholesterol versus control and for the eNampt groups treated with anti-IL-6 or anti-IL-6 + anti-IL-10 antibody versus no antibody or anti-IL-10 alone. In Experiment 1, p < 0.01 for cholesterol versus control and for the eNampt groups treated with anti-IL-6 versus control IgG. There was no statistically significant difference among the cholesterol, eNampt/cholesterol/anti-IL-6, and cholesterol/anti-IL-6 groups.