Fig 1: Adenosines promote the binding of AUF1 upon selective transcripts leading to their differential control. (A) qRT-PCR detection of selected mRNAs tested to IP with anti-AUF1 in control BMDMs activated with LPS or LPS+CGS compared to untreated (M0). Enrichment of each mRNA in AUF1-IP samples compared with its abundance in IgG-IPs and normalized to mRNA expression levels. Enrichments above two-fold were considered significant. (B) Expression levels of mRNAs bound by AUF1 were measured in extracts from BMDMs activated with LPS or LPS+CGS. (C) The half-lives (t 1/2) of Tnf, Il-12b and Vegf mRNA were quantified by measuring the time required for reducing transcript levels to 50% of their original abundance after adding actinomycin as in Figure S7B . Graphs depict mean values ± SD. Note that half-lives in Il-12b mRNAs under LPS or under LPS+CGS in the mutants lack variation since they exceed monitoring times and as such were assigned a common maximal value. In all cases, data were analyzed by two tailed, unpaired Student’s t test. *p ≤ 0.05, **p ≤ 0.01 ***p ≤ 0.001, ****p ≤ 0.0001.
Fig 2: Macrophages lacking AUF1s functions show defects in their transitions in response to adenosine receptor signals but not in response to TGFβ and IL10. Quantitation of factors marking the classical or the alternative polarization of macrophages as well as detection of hnRNPD expression in response to (A) the combination of TGFβ+IL10 and; (B) adenosine A2A receptor agonist (CGS) in LPS-activated control and M-hnRNPDΔx3,4 BMDMs (n=10). Bar graphs denote mean values ( ± SD) of secreted proteins assessed via Cytometric bead arrays; or mean fold changes (FC) in mRNAs relative to resting control values (CM0) as assessed via qRT-PCR. (C) Detection of hnRNPD, Vegfa and Thbs1 mRNAs in BALF cells from OVA-treated samples (n=5-6) relative to untreated (PBS) values as assessed via q RT-PCR. (D) Quantitation of Adora2a and Adora2b mRNAs in control and M-hnRNPDΔx3,4 BMDMs (n=10) in response to the indicated signals. Bar graphs denote mean values ( ± SD) or mean fold changes (FC) in mRNAs relative to resting control values (CM0) as assessed via qRT-PCR (*, **, ***, ****) denote p values ≤0.05, ≤0.001, ≤0.0001, ≤0.00001 respectively as determined via unpaired Student’s t-test.
Fig 3: TNF-α and IL-6 upregulation upon S100A8 and S100A9 stimulation. CD33, CD68, CD69, CD147 and TLR4 knockout ER-Hoxb8 precursor cells, as well as WT ER-Hoxb8 precursor cells were differentiated into macrophages and subsequently stimulated with LPS, S100A8 homodimer, S100A9 homodimer or incubated without stimulus (Ctrl). TNF-α and IL-6 protein levels in cell culture supernatants were quantified using LegendPlex™ assay (n = 3). Concentrations below the limit of quantification were set as LLOQ/2. Values are the means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, NS = not significant, by one-way ANOVA and Dunnett's test in comparison to Ctrl.
Fig 4: MGO modulates microglia polarization in vitro.(a) Flow cytometry analysis of iNOS expression in BV2 cells treated with LPS and different concentrations of MGO for 24 h. (b) BV2 cells were stimulated with IL-4 plus IL-13 in the presence or absence of MGO for 24 h, and the expression of Arg-1 was analyzed by flow cytometry. (c and d) Flow cytometry analysis of IL-1β and IL-6 expressions in BV2 cells treated with LPS ± 200 μM MGO for 24 h. (e) Real-time PCR analysis of IL-1β and IL-6 expressions in BV2 cells treated with LPS ± 200 μM MGO for 24 h. (f) Flow cytometry and real-time PCR analysis of TNF-α expression in BV2 cells treated with LPS ± 200 μM MGO for 24 h. (g) BV2 cells treated with LPS and 200 μM MGO for 12 or 24 h, multiplex cytokine profiling of supernatants was analyzed by cytometric bead-based immunoassay. *p < 0.05; **p < 0.01; ***p < 0.001. Ns, not significant. Three independent experiments were performed.
Fig 5: Tissue levels of pro‐inflammatory (KC, IL‐18, IL‐23, TNF‐α, IL‐1β, IL‐6) and anti‐inflammatory (G‐CSF, TARC, IL‐10) mediators from diabetic mouse model of MRSA‐infected wounds after 3 and 7 days of treatment. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; # p < 0.0001 in comparison to unloaded GG and control groups at 7 dpt.
Supplier Page from BioLegend for LEGENDplex™ MU Macrophage/Microglia Panel (13-plex) w/VbP