Fig 2: Suppression of SR-B1 in inflammatory monocytes by super-low-dose LPS is dependent on miR-24 induction.(a,b) ApoE-/- mice were pre-conditioned with PBS or super-low-dose LPS for 4 weeks together with HFD, followed by HFD feeding only for an additional 4 weeks. Total miRs isolated from splenocytes were used for real-time reversre transcriptase–PCR analyses for the relative levels of miR-24 (a) and miR-29 (b). (c) BMMs from C57 BL/6 mice were cultured with M-CSF (10 ng ml-1) in the presence super-low-dose LPS (0.1 ng ml-1) for 3 days and fresh LPS was added to the cell cultures every 2 days. Fluorescent RNA probe for miR-24-3p was added to the cell cultures 16 h before harvesting. The expression levels of miR-24-3p within CD11b+Ly6C++ monocytes were examined by flow cytometry. (d) BMMs from C57 BL/6 mice were cultured with M-CSF (10 ng ml-1) in the presence super-low-dose LPS (0.1 ng ml-1) and miR-24 antagomir was added to indicated cultures. Fresh LPS and antagomir was added to the cell cultures every 2 days. On day 5, cells were harvested and expression levels of SR-B1 within CD11b+Ly6C++ monocytes were analysed by flow cytometry. (e) A schematic illustration of the SR-B1 3'-UTR and potential miR-24-binding sites. (f,g) Luciferase activity assays in 293T cells transfected with either WT or mutant SR-B1 3'-UTR luciferase reporter plasmids in the presence of miR-24 mimic or scrambles. Quantified data are shown from cells with indicated treatment (n=3) and results are representative of three experiments. Error bars show means±s.e.m.; NS, not significant; *P<0.05, **P<0.01 and ***P<0.001; (a,b,c) Student's t-test; (d,f,g) one-way analysis of variance.

Fig 3: Myeloid cell-specific Runx2 ablation deteriorated adverse cardiac remodeling after MI. (A) Simplified scheme using Cre-loxP system is shown to generate myeloid cell-specific Runx2-deficient mice. (B, C) Seven days after MI, the sorted CD11b+ F4/80+ cells lysates from Runx2fl/fl, a control, and Runx2 CKO mice were immunoblotted with anti-Runx2 and anti-GAPDH antibodies. (B) Representative images are shown. (C) Runx2 expression was quantified. Data are shown mean ± SD (n = 4), **P < 0.01 vs Runx2fl/fl by Welch’s t-test. (D, E) Ratios of ventricular weight to tibia length (D) and lung weight to tibia length (E) were calculated 14 days after MI. Data were shown as mean ± SD (n = 11 for Runx2fl/fl mice, n = 13 for Runx2 CKO mice), *P < 0.05, **P < 0.01 vs Runx2fl/fl by Mann Whitney’s U test. (F, G) Heart sections were prepared from Runx2fl/fl and Runx2 CKO mice 14 days after MI and stained with Masson’s trichrome method. (F) Representative images are shown. (G) The ratio of fibrotic area to LV area was quantitatively estimated. Data are shown as mean ± SD (n = 11 for Runx2fl/fl mice, n = 14 for Runx2 CKO mice), **P < 0.01 vs Runx2fl/fl by Student’s t-test. (H) Quantitative RT-PCR was performed for collagen1a1, collagen3, and Tgfß1 7 days after MI. The results were normalized to that of GAPDH. Data are shown mean ± SD (n = 4 for Runx2fl/fl mice, n = 6 for Runx2 CKO mice). *P < 0.05 vs Runx2fl/fl by Mann Whitney’s U test.

Fig 4: Flow cytometric analysis of Hsp25 expression in astrocytes and microglia after LPS and TNFa treatment. Primary cortical and spinal cord mixed glial cultures were treated with 80 µg/mL LPS or 100 ng/mL TNFa. The cells were fixed, permeabilised and immunolabelled for GFAP-Cy3, CD11b-APC/Cy7 and Hsp25-AF488. (a,d) Flow cytometric analysis of cortical and spinal cord glial cultures. Left–right: Representative cytograms of untreated and LPS treated cortical glial cultures, and untreated and LPS treated spinal cord cultures. Data are presented as pseudo-colour scatter plots where blue represents low and red represents a high frequency of cells. The percent of GFAP+ve or CD11b+ve cells in each gate is provided. (b,e) The percent of Hsp25+ve astrocytes or microglia as determined from the flow cytometric analysis. (c,f) The Hsp25-AF488 fluorescent median of the astrocytic or microglial population in arbitrary units (AU). Data shown are the means ± SEM of three biological replicates. Differences between the means were determined using a two-way ANOVA followed by Bonferroni’s post hoc test, where p < 0.01 (**).

Fig 5: BM-3 17(S),18(R)-EpETE reduces the number of neutrophils. Mice were injected intraperitoneally with either commercially available Cayman (±)17,18-EpETE, BM-3 17(S),18(R)-EpETE, or vehicle only on days 0 and 5 at 30 min before DNFB application. A, On day 7, flow cytometry was used to count the number of Ly6G+ CD11b+ neutrophils and calculate their proportion of the total cell count. Data are combined from two independent experiments, and each point represents data from an individual mouse. Horizontal bars among data points indicate median values. Statistical significance was evaluated using one-way ANOVA; ***P < .001. B, Frozen ear sections obtained on day 7 were stained with fluorescein isothiocyanate-labeled Ly6G monoclonal antibody and 4',6-diamidino-2-phenylindole for immunohistologic analysis. Data are representative of two independent experiments. Bars, 100 µm