Elevated levels of CXCL2 protein in wounds of wild-type and obese mice.

<p>(a) Analysis of CXCL2 protein expression by ELISA from skin and wound tissue at different time points in wild-type and obese (<i>ob/ob</i>) mice as indicated. **, p < 0.01; (Student’s unpaired <i>t</i> test) compared to wildtype mice. Bars indicate the mean ± S.D. obtained from wounds (n = 8) isolated from four individual animals (n = 4). (b) Back wound phenotype of wildtype and ob/ob mice at day 11 upon wounding as indicated. Arrows highlight the wound areas in the wildtype mouse.</p

Expression of adipogenic transcription factors in 3T3-L1 cells.

<p>3T3-L1 pre-adipocytes were differentiated in DMI and DMII for 12 days. KLF5 (a), C/EBPα (b) and PPARγ2 (c) mRNA (upper panels) and protein (lower panels) in 3T3-L1 cells was analyzed by qRT-PCR (upper panels) or immunoblot (lower panels) at the indicated time points of differentiation. *, p < 0.05; **, p < 0.01; (Student’s unpaired <i>t</i> test) compared to non-differentiated cells. Bars indicate the mean ± S.D. obtained from four independent experiments (n = 4). Recombinant Flag-tagged KLF5 (a), C/EBPα (b) and PPARγ2 (c) served as positive controls in the immunoblots. GAPDH was used to control loading.</p

Rosiglitazone improves wound healing and subcutaneous fat cell morphology.

<p>Formalin-fixed and paraffin-embedded 13-day wound sections of mock- (a) or rosiglitazone-(b) treated mice (0.5 mg/kg/day) were assessed by AZAN trichrome staining. Collagen deposition is indicated by the blue color. The squares indicate the localizations of the respective magnifications. Scale bars are given in the photographs. Appearance of back wounds upon a 13-day mock (a) or rosiglitazone (b) administration in <i>ob/ob</i> mice is shown in the lower right panels.</p

Key transcription factors of adipogenesis are increased in subcutaneous fat of obese mice.

<p>qRT-PCR quantification of KLF5 (a), PPARγ1 (b) and PPARγ2 (c) mRNA expression in subcutaneous fat tissue of wildtype and obese (<i>ob/ob</i>) mice as indicated. *, p < 0.05 (Student’s unpaired <i>t</i> test) as compared to wildtype mice. Bars indicate the mean ± S.D. obtained from fat tissue isolated from four individual animals (n = 4). Subcutaneous fat was analyzed by immunoblot for the presence of KLF5, PPARγ1 and PPARγ2 protein as indicated (d). Lysates from differentiated 3T3-L1 adipocytes served as a positive control for KLF5 and PPAR-specific immunoblot signals. β-actin was used to control loading.</p

Effects of rosiglitazone on SAPK/JNK and NF-κB signaling in differentiated 3T3-L1 cells.

<p>3T3-L1 pre-adipocytes were differentiated in DMI and DMII for 12 days. Differentiated 3T3-L1 cells were stimulated with cytokines (25 ng/ml IL-1β, 50 ng/ml TNFα) in the presence or absence of rosiglitazone (4μM) as indicated. Cell lysates were subsequently analyzed by immunoblot for the presence of phosphorylated SAPK/JNK (a), the phosphorylation of the NF-κb p65 subunit (b) and degradation of IκBα (d). Total NF-κb p65 (c) and GAPDH (e) served as loading controls.</p

PPARγ-agonist rosiglitazone reduces Cox-2 expression in finally differentiated 3T3-L1 adipocytes.

<p>3T3-L1 pre-adipocytes remained non-differentiated in non-differentiating control medium (ND) or were stimulated to induce the differentiation process in differentiating medium (<i>DIFF</i>) for 4 days or 12 days as indicated. 3T3-L1 cells in non-differentiating medium (ND) (a) or differentiating medium (<i>DIFF</i>) (b) were then stimulated with cytokines (25 ng/ml IL-1β, 50 ng/ml TNFα) for 8 h in the presence of increasing concentrations of rosiglitazone (1–4 μM) before (day 4) and after (day 12) maturation to adipocytes. Cox-2 mRNA expression was analyzed by qRT-PCR. *, p < 0.05; n.s., not significant (Student’s unpaired <i>t</i> test) compared to cytokine-treated, but rosiglitazone-free cells. Bars indicate the mean ± S.D. obtained from four independent experiments (n = 4).</p

Altered gene expression in subcutaneous fat tissue of obese mice.

<p>qRT-PCR quantification of CXCL2 (a), CCL5 (b), leptin (c) and FABP4 (d) mRNA expression in subcutaneous fat tissue of wildtype and obese (<i>ob/ob</i>) mice as indicated. **, p < 0.01; *, p < 0.05 (Student’s unpaired <i>t</i> test) as compared to wildtype mice. Bars indicate the mean ± S.D. obtained from fat tissue isolated from four individual animals (n = 4).</p

MAPK signaling in cytokine-stimulated differentiated and non-differentiated 3T3-L1 cells.

<p>3T3-L1 pre-adipocytes were differentiated in DMI and DMII for 12 days or remained non-differentiated in basal medium I. Differentiated and non-differentiated 3T3-L1 cells were stimulated with cytokines (25 ng/ml IL-1β, 50 ng/ml TNFα) as indicated. Cell lysates were subsequently analyzed by immunoblot for the presence of phosphorylated p42/44 MAPK (a), degradation of IκBα (b) and phosphorylation of SAPK/JNK (c) as indicated. Total p42/44 (a, lower panel), β-actin (b, lower panel) or total SAPK/JNK (c, lower panel) served as loading controls.</p

Timeline diagram of the experimental procedures.

<p>Timeline diagram of the experimental procedures.</p

DE leads to a prolongation of the dTT in linear correlation with DE plasma concentrations (Hemoclot™ assay).

<p>A) dTT was measured with the Hemoclot™ assay in anticoagulation-naïve mice (n = 4), mice receiving 3×37.5 mg/kg over a 24 h feeding period (n = 3) and mice receiving 3×75 mg/kg (n = 3). Statistical significance was assessed with one-way ANOVA and Bonferroni correction. B) Calibration of the coagulometer with lyophilized standard DE plasma gives the DE concentration from the dTT values. Statistical significance was assessed with one-way ANOVA and Bonferroni correction. ** p<0.01; *** p<0.001.</p