Fig 1: Chronic Over-nutrition Reduces Wnt/ß-Catenin and Ppar? Pathway Activation in VAT-cDCsZbtb46-GFP+ mice were fed a chow or WD for 12 weeks.(A) mRNA expression of Wnt proteins was analyzed by RT-PCR in mice on WD compared to chow diet (12 weeks). Error bars indicate the geometric mean of five biological replicates.(B) Wnt10b protein levels were detected in VAT homogenates (n = 3).(C) mRNA expression of Wnt10b in Ctnnb1-/- and Ppar?-/- mice (n = 10).(D) Western blot analysis of active ß-catenin in purified CD103+ Zbtb46-GFP+ cDCs from chow- and WD-fed mice.(E and F) cDC purified from spleen and VAT from Zbtb46-GFP mice were incubated overnight with 5 µg GLA in the presence of ß-catenin agonist (SB) or DMSO as control. IL-6 (E) and IL-10 (F) production was measured in supernatants. Bars represent mean ± SD and are representative of two independent experiments (n = 6).(G) Western blot analysis of PPAR? in purified CD11b+ Zbtb46-GFP+ cDCs from chow- and WD-fed mice.(H and I) VAT-cDCs were purified from chow- and WD-fed Zbtb46-GFP+ mice. Cells were cultured overnight with PPAR? agonist (RGZ), and CD36 expression was evaluated by flow cytometry (H). Histograms are representative of three independent experiments (H) (n = 3) and mRNA expression of CD36 and other downstream genes (I) (n = 5).(J and K) As described in (E) and (F) but cells were cultured with PPAR? agonist overnight and IL-6 (J) and IL-12 (K) were measured in supernatants. Bars represent mean ± SEM and are representative of two independent experiments. Statistical analysis was performed with Student’s test, *p < 0.05, **p < 0.01, and ***p < 0.0005.See also Figure S6.
Fig 2: Activation of the Wnt/ß-Catenin and PPAR? Pathway in cDC1 and cDC2 DCs, Respectively, Suppresses TLR4-Induced InflammationcDC1 and cDC2 were purified from spleen and VAT by cell sorting as CD11chi MHCII+ GFP+ cells.(A and B) Cells were incubated overnight with 5 µg GLA in the presence of ß-catenin (SB) or PPAR? (RGZ) agonist or DMSO control. IL-6 (A) and IL-10 (B) production in supernatants was measured by ELISA. Bars represent the mean ± SEM and are representative of three independent experiments (n = 9).(C) A total of 300 µg VAT was isolated from lean mice and cultured overnight (Sup) or immediately lysed (Lys), and Wnt10b production was analyzed by ELISA. Bars represent the mean ± SEM (n = 3).(D) Total GFP+ cDCs were purified and cultured overnight with 100 ng/mL recombinant Wnt10b or SB agonist as positive control; IL-10 was determined in the supernatant by ELISA (n = 9).(E and F) As described in (D), but in addition, cells were stimulated with GLA in the presence of recombinant Wnt10b; the production of IL-6 (E) and IL-10 (F) was determined in the supernatant by ELISA. Bars represent the mean ± SEM and are representative of three independent experiments (n = 9). Statistical analysis was performed with Student’s test, *p < 0.05, **p < 0.01, and ***p < 0.0005.
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