Tissue factor-par2 signaling promotes diet-induced obesity and adipose
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1 Supplementary figures for Tissue factor-par2 signaling promotes diet-induced obesity and adipose inflammation. Leylla Badeanlou 1, Christian Furlan-Freguia 2, Guang Yang 1, Wolfram Ruf 2,3, and Fahumiya Samad 1,3 1 Department of Cell Biology, Torrey Pines Institute for Molecular Studies, San Diego, CA, USA. 2 Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA, USA. 3 Correspondence to Wolfram Ruf, ruf@scripps.edu and Fahumiya Samad, fsamad@tpims.org 1
2 Supplementary Figure 1: Regulation of body mass by TF-PAR2 signaling. (a) TF-PAR2 signaling regulates body weights on a normal diet. Body weights of week old male mice on a low fat chow diet showed no effect of PAR1-deficiency (F2r -/- mice) versus WT controls. F2rl1 -/-, TF ΔCT, and TF ΔCT /F2rl1 -/- mice weighed significantly less than WT mice; mean ± SD, n = 6-12; *** P < for WT versus knock-out mice. (b) Reduced adipose tissue mass in HFD F2rl1 -/- and TF ΔCT mice. Mice were sacrificed after 16 weeks on a HFD and organ weights were normalized to body weights. Mean ± SD, n = 6-12; * P < 0.05 for WT versus knock-out mice. 2
3 Supplementary Figure 2: Glucose homeostasis in WT and knock-out mice. (a, b) Glucose tolerance and insulin tolerance tests for WT or TF ΔCT mice on a HFD for 16 weeks; mean ± SD, n = 15; *P <0.05, **P<0.01, ***P<0.001 WT versus TF ΔCT. (c, d) Glucose tolerance and insulin tolerance tests of WT, F2rl1 -/-, and TF ΔCT /F2rl1 -/- mice on a HFD; mean ± SD, n= 8; # P<0.05, ## P<0.01, ### P<0.001 for WT versus TF ΔCT /F2rl1 -/- *P<0.05, **P<0.01, ***P<0.001 for WT versus F2rl1 -/-. 3
4 Supplementary Figure 3: TF expression in macrophages from BM transplanted mice. Lethally irradiated WT mice were reconstituted with BM from WT, TF ΔCT or F2rl1 -/- mice. After 6 weeks of recovery, mice were fed a HFD for at least 16 weeks and SVF cells were obtained by collagenase digestion of the epididymal VAD. (a) Gates of CD11b + /F4/80 + and CD11b + /CD11c + macrophages in the SVF fraction from WT mice transplanted with the indicated BM. (b) TF mean fluorescence of CD11b + /CD11c + cells from chimeras with the indicated BM; mean ± SD, n = 3. 4
5 Supplementary Figure 4: Improved glucose homeostasis in mice with a TF-PAR2 signalingdeficient BM compartment. Glucose tolerance (a) and insulin tolerance (b) tests in WT mice BM transplanted with WT, TF ΔCT or F2rl1 -/- BM after 16 weeks on a HFD; mean ± SD, n = 10-14, *P<0.05, **P<0.01 for WT versus knock-out BM chimeras. 5
6 Supplementary Figure 5: Control experiments for TFKI BM chimeras and antibody 21E10 therapy. HFD-induced weight gain (a) and insulin tolerance test (b) of chimeric WT mice reconstituted with BM from WT (n = 7) or human TF knock-in (TFKI) mice; mean ± SD, n = 12. (c) Insulin tolerance test in TF ΔCT mice on a HFD for 16 weeks was performed 24 hours after injection of 20 mg per kg body weight of antibody 21E10 to mouse TF or control antibody; mean ± SD, n = 4. 6
7 Supplementary Figure 6: Contribution of TF-PAR2 signaling to metabolism and energy expenditure. Food intake, activity, O 2 consumption and CO 2 output, and respiratory exchange ratio (RER) in WT versus TF ΔCT (a) or F2rl1 -/- (b) mice after 16 weeks on a HFD. Note that the presented data in these panels and in Fig. 4 of the main manuscript were not normalized for body weights. Normalization for body weights showed that both, TF ΔCT and F2rl1 -/- mice (c) as well as BM chimeras of these strains carrying WT BM (d) have significantly increased VO 2 and VCO 2 relative to WT controls; mean ± SD, n = 5; *P<0.05, **P < 0.01 different from WT control. With the understanding that such normalization may underestimate energy expenditure of more obese WT mice, these data indicate that TF ΔCT and F2rl1 -/- mice are protected from obesity due to a common mechanism of increased energy expenditure. 7
8 Supplementary Figure 7: Specificity of antibody 21E10 for mouse TF. Rat monoclonal antibody 21E10 was generated by immunizing rats with recombinant mouse TF extracellular domain and screening for reactivity with mouse, but not human TF. Antibody 21E10 dose-dependently inhibited TF-VIIa mediated FX activation by mouse adipocytes (left panel), but did not inhibit Xa generation by relipidated recombinant human TF (right panel). Inhibition with anti-human TF antibody 5G9 is shown as a positive control; mean ± SD, n = 3. 8
Supplementary Figure 1
Supplementary Figure 1 how HFD how HFD Epi WT p p Hypothalamus p p Inguinal WT T Liver Lean mouse adipocytes p p p p p p Obese mouse adipocytes Kidney Muscle Spleen Heart p p p p p p p p Extracellular
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1 Supplemental methods cute insulin challenge: For assay of biochemical responses to insulin stimulation, we 3 4 6 7 8 9 1 anesthetized mice after a 6- h fast. We ligated vessels supplying one side of
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