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. 2012 May;32(10):2010–2019. doi: 10.1128/MCB.06193-11

Fig 4.

Fig 4

JNK and TLR4 at least partially mediate the effects of RBP4 on insulin stimulation and cytokine production. (A) Western blot of total and phosphorylated Akt in 3T3L1 adipocytes from direct contact coculture with RAW264.7 macrophages. Cells were pretreated with JNK or IKK inhibitor (1 or 10 μM for 30 min), and then human holo-RBP4 was added (50 μg/ml for 24 h) prior to insulin (100 nM for 10 min). (B) Quantification of experiments shown in panel A. All conditions were normalized to cells treated with PBS and then stimulated with insulin. Data are means ± SE. *, P < 0.05 versus insulin-stimulated (+) with no RBP4. (C) mRNA expression of TNF-α, IL-6, and MCP-1 in RAW264.7 macrophages treated with JNK inhibitor (1 or 5 μM) and RBP4 as in panel A. The control for RBP4 was dialysate, and the control for JNK inhibitor was dimethyl sulfoxide (DMSO). AU, arbitrary units. Data are means ± SE and normalized to GAPDH expression (n = 3 to 4). *, P < 0.05 versus no RBP4; #, P < 0.05 versus no JNK inhibitor; +, P < 0.05 versus RBP4 plus 1 μM JNK inhibitor, one-way ANOVA. In, inhibitor. (D) TNF-α, IL-6, and MCP-1 secretion from primary mouse macrophages stimulated with holo- or apo-RBP4 or add-back RBP4 (50 μg/ml for 24 h). Macrophages were harvested from either control mice with functional JNK1 and JNK2 or mice with JNK1 and JNK2 deleted (JNK1/2 KO). Data are means ± SE (n = 4). *, P < 0.05 versus vehicle (dialysate) treatment within the same genotype; #, P < 0.05 for JNK1/2 KO versus control with same treatment; +, P < 0.05 versus holo-RBP4 and add-back RBP4 treatment within same genotype by two-way ANOVA with post hoc analysis. (E) TNF-α and IL-6 secretion from primary mouse macrophages stimulated with human holo- or apo-RBP4 or add-back RBP4 (50 μg/ml for 24 h). Macrophages were harvested and pooled from either two wild-type mice (open circles) or two mice with TLR4 deleted (TLR4 KO) (closed circles), and the experiments were done in duplicate. The bars show the mean with individual values indicated by circles (n = 2). *, P < 0.05 versus vehicle (dialysate) treatment within the same genotype; #, P < 0.05 for TLR4 KO versus control with same treatment; +, P < 0.05 versus holo- and add-back RBP4 treatment within same genotype by two-way ANOVA with post hoc analysis.