Fig 1: Reproductive ageing is associated with a local inflammatory microenvironment in the mouse ovary. Gene expression levels of Tnfa (A), Il6 (B), Il1a (C) and Il1b (D) in ovaries from 2, 6, 12 and 18-month-old mice. n = 6 per cohort. Data are presented as mean ± SEM; (A) and (C): Kruskal–Wallis test, Dunn’s multiple comparisons test: a, b and c are significantly different among groups (p < 0.05); (B) and (D): Ordinary One-way ANOVA, Tukey’s multiple comparisons test: a and b are significantly different among groups (p < 0.05). (E) Representative image of a Western blot showing ovarian protein levels of TNF-a (precursor), TNF-a (mature), IL-6 and IL-1ß from 2, 6, 12 and 18-month-old mice. Quantification of protein levels of TNF-a (precursor) (F), TNF-a (mature) (G), IL-6 (H) and IL-1ß (I) in ovaries from 2, 6, 12 and 18-month-old mice. n = 5 per cohort. Data are presented as mean ± SEM; Mann–Whitney test (*p < 0.05). Each dot represents one animal. mRNA levels of Csf1 (J), Csf2 (K), Ccl5 (L) and Il10 (M) in ovaries from 2, 6, 12 and 18-month-old mice. n = 6 per cohort. Data are presented as mean ± SEM; I: Ordinary One-way ANOVA, Tukey’s multiple comparisons test: a and b are significantly different among groups (p < 0.05); (J),K,L): Kruskal–Wallis test, Dunn’s multiple comparisons test: a, and b are significantly different among groups (p < 0.05).
Fig 2: The expression of pro-inflammatory genes is reduced in ovaries from aging Asc-/- mice. Gene expression levels of Tnfa (A), Il1a (B), Il1b (C), Il6 (D), Il10 (E), Csf1 (F), Csf2 (G), and Ccl5 (H) in ovaries from 2, 6, 12, and 18-month-old WT, Asc-/-, and Nlrp3-/- mice. n = 3–6 per cohort. All data are presented as mean ± SEM. For each age group, comparisons were made with WT using Student's t-test (A–H) or Mann-Whitney test (A–H) (*p < 0.05, **p < 0.01, ***p < 0.001).
Fig 3: Models for the negative regulation of NF-?B signaling pathways by caspase-3-mediated cleavage of NF-?Bs.A In mammals, apoptotic effector caspase-3 cleaves of NF-?B members p65/Rel A, RelB, and c-Rel resulting in a comprehensive repression of both NF-?B signaling pathways, which is proposed to act as a self-protective mechanism to prevent overreactive innate immune responses. B In Drosophila, apoptosis mediates the proteolysis of DIF, the NF-?B of Toll pathway, resulting in the repression of Toll immune signaling, dependently of the effector caspases drICE and DCP-1.
Fig 4: DrICE and DCP-1 cleave DIF in Drosophila.A At 6 h post M. luteus challenge, total RNA extracts were prepared from adult flies with the indicated genotypes and treatments, and subjected to qRT-PCR of Drs mRNA normalized by Rp49. Each group contains 10 female flies and 10 male flies. B Quantitative RT-PCR analysis of Drs mRNA in S2 cells transfected with HA-tagged Toll?LRR, together with drICE?N or DCP1?N or treatment with TG. C Quantitative RT-PCR analysis of Drs mRNA in S2 cells transfected with HA-tagged Toll?LRR together with indicated dsRNAs. Total RNA extracts were prepared and detected via Northern blots using the indicated probes. D Quantitative RT-PCR analysis of Drs mRNA in S2 cells transfected with plasmids as indicated. E Immunoblot of purified WT and D212A mutant of DIF proteins incubated without (-) or with purified drICE?N (+) for 60 min at 37 °C. The blot was probed with anti-Flag antibody. F, G Quantitative RT-PCR analysis of Drs mRNA in S2 cells transfected with plasmids as indicated. H The S2 cells were ectopically expressed with the indicated proteins for 48 h. Co-immunoprecipitation were performed by using anti-HA or anti-Flag antibody, followed by immunoblot with indicated antibodies.
Fig 5: Agomir-IL-35 in murine RAW264.7 macrophages prevents LPS-induced inflammation. All data are shown as mean ± standard error of mean (n=6, per group). ***P<0.001 vs. control; ###P<0.001 vs. LPS; †††P<0.001 vs. agomir-NC; £££P<0.001 vs. agomir-LPS. IL-35, interleukin-35; LPS, lipopolysaccharide; con, control; NC, negative control; TNF, tumor necrosis factor.
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