Fig 1: miR29c mediates the effects of NOD2 in limiting production of regenerative factors in ECs and DCs(A) Thymuses were isolated from 6- to 8-week C57BL/6 WT or Nod2-/- mice at day 3 following SL-TBI, and the expression of 3p and 5p arms of miR29a, miR29b, or miR29c was analyzed by qPCR (3p, n = 4/group; 5p, n = 7/group across two independent experiments).(B) DPs, ECs, and DCs were FACS purified from WT thymuses at day 0 or 3 after SL-TBI and expression of miR29c-5p was analyzed by qPCR (DCs, n = 4; ECs/DPs, n = 6–7/population/time point across two independent experiments).(C) Thymic exECs were generated as previously described (Seandel et al., 2008; Wertheimer et al., 2018) and transfected with a miR29c mimic. 20 h after transfection, the expression of Bmp4 was analyzed by qPCR (n = 7 independent experiments).(D) exECs were transfected with a miR29c inhibitor, and the expression of Bmp4 was analyzed by qPCR 20 h after transfection (n = 4 independent experiments).(E) CD11c+ DCs were isolated from untreated C57BL/6 thymuses and transfected with a miR29c mimic. 20 h after transfection, Il23p19 was analyzed by qPCR (n = 5 across three independent experiments).(F) HEK293 cells were co-transfected with either Bmp4-3' UTR, Il12p40-5' UTR, or Il23p19-3' UTR luciferase constructs and a miR29c-5p mimic. Binding activity was quantified by measuring luciferase activity after 20 h (n = 5–7 independent experiments). Graphs represent mean ± SEM; each dot represents a biologically independent observation. See also Figure S3.
Fig 2: ELISA and immunohistochemistry confirm gene expression data in Fig. 6. Ldlr-/- mice were fed the four diets, and the levels of protein in jejunum were determined by ELISA or the number of goblet cells or Paneth cells in jejunum were determined by immunohistochemistry as described in Materials and methods. The gender, age, and number of mice per group for each panel are shown in supplemental Table S5. A: IL-36?. B: IL-23. C: IL-22. D: NOTCH2. E: DLL4. F: ATOH1. G: GFI1. H: Goblet cells/crypt. I: Goblet cells/villus axis. J: Paneth cells/crypt. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Fig 3: The mRNA levels of inflammatory cytokines in the skin lesion treated with different preparations. (A) Il-23, (B) Il-17A, (C) Tnf-a, (D) Il-1ß, (E) Il-6. *P < 0.05, **P < 0.01, ***P < 0.001 compared with IMQ group, #P < 0.05, ##P < 0.01 compared with indicated group. Each value represents the mean and SE (n = 6).
Fig 4: L-THE downregulates the expression of inflammatory genes. (A,B) Volcano plot. (A) showed the expression of inflammatory response associated genes, and heatmap. (B) showed the significantly differentiated genes about interleukin, CXCL, and CCL family genes based on RNA-seq data from IMQ-induced psoriasis mice treated with 100 mM L-THE or H2O for 5 days. (C) RT-PCR analysis of the mRNA levels of IL-23p19, TNF-a, CXCL2, and S100A8 in IMQ-induced psoriasis mice (n = 6 per group) treated with 100 mM L-THE or H2O for 0 or 5 days. (D) ELISA analysis of IL-23A in IMQ-induced psoriasis mice (n = 6 per group) treated with 100 mM L-THE or H2O for 0 or 5 days. Data is representative of three independent experiments. p values are determined by two-way ANOVA. ***p < 0.001.
Fig 5: TAM receptor detection of phosphatidylserine mediates thymocyte suppression of regenerative factors(A and B) Thymocytes were isolated from untreated C57BL/6 mice and incubated for 4 h with Dexamethasone (100 nM) or zVAD-FMK (20 µM). After 4 h, apoptotic thymocytes (ACs) were washed and co-cultured with exECs (A) or freshly isolated CD11c+ DCs (B) for 24 h after which Bmp4 expression was analyzed by qPCR (n = 7 across 5 independent experiments) or IL-23 was analyzed by intracellular cytokine staining (n = 5 across two independent experiments).(C–G) 6- to 8-week-old C57BL/6 mice were given sublethal TBI (550 cGy), and the thymus was harvested at days 0, 1, 2, 3, and 7. (C) Annexin V staining on DP thymocytes (displayed are concatenated plots from 3 individual mice, representative of three independent experiments). (D) Staining for CD4 and CD8 on thymus cells (displayed are concatenated plots from 3 individual mice, representative of three independent experiments). (E) Total number of DP thymocytes (solid line; left axis) compared with proportion of Annexin V+ DP thymocytes (red broken line; right axis) (n = 8 across 3 independent experiments). (F) Absolute binding of Annexin V in the thymus (n = 8 across 3 independent experiments). (G) Correlation of absolute thymic binding of Annexin V with total number of cells in the thymus (n = 3/time point comprising one of three independent experiments).(H) DP thymocytes, DCs, or ECs were FACS purified from untreated C57BL/6 mice, and expression of Axl, Mer, and Tyro3 was analyzed by qPCR (DP, n = 7; DC, n = 5; EC, n = 7 across two independent experiments).(I and J) Thymocytes were isolated from untreated C57BL/6 mice and incubated for 4 h with dexamethasone (100 nM). After 4 h, apoptotic thymocytes (ACs) were washed and co-cultured with exECs (I) or freshly isolated CD11c+ DCs. (J) in the presence or absence of the TAM receptor inhibitor RXDX-106 (25 µM) for 20 h after which Bmp4 expression was analyzed by qPCR (n = 6/treatment across 2 independent experiments) or IL-23 was analyzed by intracellular cytokine staining (n = 4). Graphs represent mean ± SEM; each dot represents a biologically independent observation. See also Figure S4.
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