Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: MEK1/2 Inhibition Promotes Macrophage Reparative Properties 1

doi: 10.4049/jimmunol.1601059

Figure Lengend Snippet: BMDM M2 gene expression is increased during IL-4/IL-13 polarization by the MEK1/2 inhibitor PD0325901. (A) One of three representative experiments showing constitutive expression of pERK1/2 in resting (M0) and IL-4/IL-13 treated conditions in murine BMDM. There was early reduction of pERK1/2 at 15-60 minutes post-stimulation with MEKi (PD0325901). (B) One of three representative experiments showing constitutive expression of pERK1/2 in resting (M0) and IL-4 treated human MDM. Western blots of protein lysates from 0 and 48 hours show decreased pERK1/2 after PD0325901 treatment. (C) M0 and IL-4/IL-13-treated BMDM exposed to carrier control or MEKi over 48 h were processed for qRT-PCR to determine relative quantification (RQ) of Retnla, Ym1, Ccl17, Tgfb1, and Arg1 mRNA normalized to time-matched M0 + carrier control samples. Treatment with MEKi led to a significant increase in IL-4/IL-13 dependent gene expression of Retnla, Ym1, Ccl17, Tgfb1, but not Arg1. Data from 3-4 biological replicates (mean±SEM). (D) At 48 h, IL-4/IL-13 treated cells exposed to carrier control or MEKi were processed for surface staining of CD71 and CD206. Change in mean fluorescent intensity (ΔMFI) was determined by subtracting the MFI of the isotype control samples from that of each antibody. MEKi treatment led to increased surface expression of both CD71 and CD206. Data mean ± SD are from triplicate samples from one representative experiment of two. (E) LPS-treated BMDM were treated with IL-4/IL-13 + carrier or PD0325901. At 48 h, MEKi treatment led to increased expression of Retnla, Ym1, and Tgfb1. Data are mean ± SD from 6 independent experiments. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Article Snippet: Validated TaqMan FAM primer probes for the murine genes Retnla Mm00445109, Hprt M m01545399, Ym1 Mm00657889, Tgfb1 Mm00441724 Ccl17 Mm01244826, Arg1 Mm00475988, Mertk Mm00434920, Abca1 Mm00442646, PU.1 Mm00488140, IL10 Mm00439614 and human genes Hprt1 Hs02800695, Tgm2 Hs00190278, Tgfb1 HS00998133, Mrc1 HS00267207, and Ccl17 HS00171074 were purchased from Life Technologies (Carlsbad, CA).

Techniques: Gene Expression, Expressing, Western Blot, Control, Quantitative RT-PCR, Quantitative Proteomics, Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: MEK1/2 Inhibition Promotes Macrophage Reparative Properties 1

doi: 10.4049/jimmunol.1601059

Figure Lengend Snippet: Efficacy of MEK1/2 pathway inhibition results in differential enhancement of BMDM IL-4/IL-13 polarization. BMDM were stimulated with IL-4/IL-13 with the addition of media (control), DMSO (+ carrier), PD0325901, U0126, or PD98059 for 48 hours. Cells were processed for RNA and protein. (A) Relative quantification (RQ) of Retnla gene expression normalized to M0 showing the greatest effect of PD0325901 (mean ± SD of 4 biological replicates). (B) BMDM protein lysates were probed for ERK1/2 phosphorylation (pERK1/2), ERK1/2, Relmα, and β-actin and quantified using densitometry. (C) Relative quantification of the ratio of pERK1/2:ERK1/2 expressed as percent of IL-4/IL-13 + carrier controls showing the greatest effect of PD0325901. (D) Ratio of Relmα:β-Actin also demonstrating the greatest effect of PD0325901. (E) BMDM protein lysates from LysMCre+/+MEK1fl/fl BMDM (LysMCre+/+) compared to LysMCre−/−MEK1fl/fl controls (LysMCre−/−) demonstrating a reduction in MEK1 protein from LysMCre+/+ cells. Lane 1: M0 + Carrier, Lane 2: IL-4/IL-13 + Carrier, Lane 3: IL-4/IL-13 + MEKi. (F) Relative quantification of Retnla gene expression from IL-4/IL-13 treated LysMCre+/+ and LysMCre−/− BMDM normalized to M0 (mean ± SD of triplicate samples). Data are from one of two representative experiments. (G) BAL alveolar macrophages were IL-4/IL-13 polarized with the addition of either DMSO (+ carrier) or PD0325901 (+MEKi) ex vivo for 48 hours. qRT-PCR was used to measure the relative quantification (RQ) of Retnla, Ym1, Tgfb1, and Ccl17. Data are ± SD of the fold increase in PD0325901 compared to carrier. (n=7 biological replicates for each group collected from 2 independent experiments.) Statistical comparisons are versus carrier-treated samples or as indicated. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Article Snippet: Validated TaqMan FAM primer probes for the murine genes Retnla Mm00445109, Hprt M m01545399, Ym1 Mm00657889, Tgfb1 Mm00441724 Ccl17 Mm01244826, Arg1 Mm00475988, Mertk Mm00434920, Abca1 Mm00442646, PU.1 Mm00488140, IL10 Mm00439614 and human genes Hprt1 Hs02800695, Tgm2 Hs00190278, Tgfb1 HS00998133, Mrc1 HS00267207, and Ccl17 HS00171074 were purchased from Life Technologies (Carlsbad, CA).

Techniques: Inhibition, Control, Quantitative Proteomics, Gene Expression, Phospho-proteomics, Ex Vivo, Quantitative RT-PCR

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: MEK1/2 Inhibition Promotes Macrophage Reparative Properties 1

doi: 10.4049/jimmunol.1601059

Figure Lengend Snippet: MEKi increases BMDM STAT6 pathway activation during IL-4/IL-13 stimulation. (A) BMDM protein lysates collected at 1, 4, 24, and 48 hours after stimulation of M0 + Carrier and IL-4/IL-13 + Carrier or + MEKi. Blots were probed for pSTAT6, STAT6, SOCS1, SOCS3, and β-Actin one representative experiment of n=4-6. (B) Densitometry quantitation of the ratio of pSTAT6/STAT6, SOCS1/Actin, and SOCS3/Actin normalized to carrier-treated samples demonstrating increased pSTAT6 and reduced SOCS1 and SOCS3 proteins in MEKi treated samples. (C, D) BMDM from wild-type (WT) or Stat6−/− Balb/c mice were stimulated with IL-4/IL-13 with the addition of DMSO (+ Carrier) or 0.5 μM PD0325901 (+MEKi). (C) At 48 h, Retnla, and Ym1 mRNA expression was measured and expressed as fold change (RQ) relative to respective M0 conditions. There was marked reduction in both Retnla and Ym1 in Stat6−/− compared to WT cells. Data are from 3-5 biological replicates and show the mean for each sample comparing matched carrier and inhibitor treated samples. (D) Protein lysates were collected at serial time points after stimulation. Relmα, STAT6 and β-Actin were detected by western blot. There was no detectable STAT6 or Relmα proteins in Stat6−/− cells compared to that of WT. Blots are from one representative experiment of three. (E) BMDM were stimulated with IL-4/ IL-13 with the addition of carrier or MEKi. RNA was collected over 48 h to determine the relative quantification (RQ) of PU.1 normalized to time-matched M0 conditions. MEKi treatment led to increases in PU.1 mRNA starting at 12 h (mean of 3-4 independent experiments). *P<0.05, **P <0.01.

Article Snippet: Validated TaqMan FAM primer probes for the murine genes Retnla Mm00445109, Hprt M m01545399, Ym1 Mm00657889, Tgfb1 Mm00441724 Ccl17 Mm01244826, Arg1 Mm00475988, Mertk Mm00434920, Abca1 Mm00442646, PU.1 Mm00488140, IL10 Mm00439614 and human genes Hprt1 Hs02800695, Tgm2 Hs00190278, Tgfb1 HS00998133, Mrc1 HS00267207, and Ccl17 HS00171074 were purchased from Life Technologies (Carlsbad, CA).

Techniques: Activation Assay, Quantitation Assay, Expressing, Western Blot, Quantitative Proteomics

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: MEK1/2 Inhibition Promotes Macrophage Reparative Properties 1

doi: 10.4049/jimmunol.1601059

Figure Lengend Snippet: MEKi promotes in vivo macrophage efferocytosis and M2 polarization. (A) Mice received either carrier or MEKi 24 h prior to intraperitoneal delivery of apoptotic neutrophils. Additional control mice did not receive PMNs. Mice were subjected to peritoneal lavage and cytospin preparations were made from recovered cells and stained with Diff-Quick. The percent of macrophages with ingested cells was quantified and data from three independent experiments are shown. MEKi treatment led to a significant increased in efferocytosis. (B-E) In a separate in vivo model of lung injury, mice received oropharyngeal delivery of LPS on day 0 and MEKi on days 1 and 3 post-LPS. Mice were monitored for weight change (B) and euthanized on days 2 and 4 for assessment of BAL cell counts and differential (C,D). On day 4, alveolar macrophages were isolated for assessment of M2 gene and protein expression (E). (B) Initial weight loss was similar between MEKi and carrier control groups, and MEKi treated mice had faster recovery of their weights starting at day 2. Day 2 (n=26-27/condition), Days 3-4 (n=16/condition). (C,D) On days 2 and 4, there were fewer BAL total cells due to reduced numbers of neutrophils in MEKi treated mice. Macrophage numbers were similar in both groups. (n=5-6 mice/group; 2-3 experimental replicates). (E) On day 4, alveolar macrophages from MEKi treated mice had greater expression of Ym1 and Ccl17 mRNA (normalized to carrier control) and greater expression of CD71 as measured by FACS. (n=10-12/group). *P<0.05, **P<0.01, ***P<0.001, ***P<0.0001.

Article Snippet: Validated TaqMan FAM primer probes for the murine genes Retnla Mm00445109, Hprt M m01545399, Ym1 Mm00657889, Tgfb1 Mm00441724 Ccl17 Mm01244826, Arg1 Mm00475988, Mertk Mm00434920, Abca1 Mm00442646, PU.1 Mm00488140, IL10 Mm00439614 and human genes Hprt1 Hs02800695, Tgm2 Hs00190278, Tgfb1 HS00998133, Mrc1 HS00267207, and Ccl17 HS00171074 were purchased from Life Technologies (Carlsbad, CA).

Techniques: In Vivo, Control, Staining, Diff-Quik, Isolation, Expressing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: HSP70-binding motifs function as protein quality control degrons

doi: 10.1007/s00018-022-04679-3

Figure Lengend Snippet: The APPY peptide functions as a degron in human cells. a Upon transfection of the HEK293T landing pad cells, BxbI catalyzes site-specific recombination between BxbI attachment sites in the vector and the landing pad, leading to single-copy expression of GFP-APPY from the Tet-on promoter. As the vector also contains an internal ribosomal entry site (IRES) followed by mCherry, the GFP-APPY levels can be normalized to the mCherry levels. 2A signifies self-cleaving peptides. b GFP:mCherry ratio distributions of cells expressing the indicated GFP-APPY variants, obtained by flow-cytometry of ~ 10,000 cells. c GFP:mCherry ratio distributions of cells expressing the GFP-APPY (RLLL) peptide or the DAAA variant of APPY after 5 h treatment with 15 μM bortezomib (BZ) or DMSO (solvent control), obtained by flow-cytometry of ~ 10,000 cells. d GFP:mCherry ratio distributions of cells expressing the APPY (RLLL) peptide or the DAAA variant of APPY after 22 h treatment with 5 μM YM01 or no treatment, obtained by flow-cytometry of ~ 10,000 cells. e Relative expression of the HSPA1A and HSPA1B genes derived from qPCR. The results were normalized to the expression of non-recombinant cells. n = 3, error bars show the standard deviation, asterisks indicate significant differences (p < 0.05) based on a two-tailed unpaired Student’s t test

Article Snippet: HEK293T drug treatments and flow cytometry profiling For the YM01 treatment cells were treated with 5 μM of YM01 (StressMarq Biosciences) for 22 h before flow-cytometry profiling.

Techniques: Transfection, Plasmid Preparation, Expressing, Flow Cytometry, Variant Assay, Solvent, Control, Derivative Assay, Recombinant, Standard Deviation, Two Tailed Test

Journal: Epilepsia

Article Title: Complex alterations in microglial M1/M2 markers during the development of epilepsy in two mouse models.

doi: 10.1111/epi.12960

Figure Lengend Snippet: Figure 1. Microglial-specific expression of M1 and M2 markers peak at the acute (3 days post-SE) but not early chronic (21 days post-SE) time point in pilocarpine SE forebrains as determined by flow cytometry. (A) M1-associated cytokines were collectively increased at the 3-day time point in SE compared to No-SE animals, including TNFa, IL-1b, IL-6, and IL-12, but returned to control levels after 21 days. (B) M2- associated cellular markers, Arg1, Ym1, IL-4, and IL-10 were all increased 3 days, but not 21 days, after SE. Only Ym1 remained elevated in SE mice at 21 days post- SE. (C) Representative median fluorescent intensity (MFI) curves of M1/M2 marker expression on microglial cells comparing naive (dotted line), No-SE (solid line, open), and SE (shaded fill) animals determined by flow cytometry at the 3-day post-SE time point. Data shown are normalized to naive controls (n = 8 per group for 3 days; n = 12 per group for 21 days after SE). ***p < 0.001, ****p < 0.0001. Mean fold change SEM shown. Two-way analysis of variance (ANOVA) with Bonferroni posttest used for statistical analysis of all data sets. Epilepsia ILAE

Article Snippet: Intracellular Arg1 (Fluorescein, IC5868F; R&D Systems, Minneapolis, MN, U.S.A.) and Ym1 (nonconjugated, MAB2446; R&D Systems; secondary – PE conjugated) were visualized after cell permeabilization.

Techniques: Expressing, Cytometry, Control, Marker

Journal: Epilepsia

Article Title: Complex alterations in microglial M1/M2 markers during the development of epilepsy in two mouse models.

doi: 10.1111/epi.12960

Figure Lengend Snippet: Figure 2. mRNA expression of M1 and M2 phenotypic markers at different stages throughout the pilocarpine model in forebrain (acute 3 days post-SE) and the hippocampal formation (early and late chronically epileptic time points) largely peak early after SE. (A) M1-associated markers TNFa, IL-1b, CD16, and CD11b and the astrocytic marker GFAP were significantly increased in forebrain mRNA of SE animals compared to No-SE controls at the acute post-SE time point. At the early chronic time point, IL-1b, CD11b, and GFAP remained elevated above No-SE control levels in the hippocampal formation, despite there being no obvious changes in whole cortical samples (data not shown). TNFa mRNA levels, however, were significantly reduced at the early chronic stage in hippocampus. No significant changes to any M1 markers were observed in hippocampal mRNA extracts in the late chronic phase (n = 5–9 per group). (B) M2-associated markers Arg1, Ym1, and IL-4 were significantly increased in SE animals compared to No-SE controls, whereas FIZZ-1 and CD206 showed signifi- cant reductions in expression in SE animals at the acute time point in forebrain (n = 5–9 per group). Together this indicates a heteroge- neous M1/M2 microglial activation state acutely after pilocarpine-induced SE. M2-associated Ym1 was the only marker for which mRNA amounts decreased in the hippocampal formation at the early chronic time point (n = 12 per group). No significant changes to any other markers were found in either the early or late chronic phases in hippocampal mRNA extracts. Data were expressed relative to house- keeping gene mRNA expression. Mean SEM shown for each data set. **p < 0.01, ***p < 0.001, ****p < 0.0001. Independent unpaired student’s t-tests used for statistical analysis of each data set. Epilepsia ILAE

Article Snippet: Intracellular Arg1 (Fluorescein, IC5868F; R&D Systems, Minneapolis, MN, U.S.A.) and Ym1 (nonconjugated, MAB2446; R&D Systems; secondary – PE conjugated) were visualized after cell permeabilization.

Techniques: Expressing, Marker, Control, Activation Assay

Journal: Epilepsia

Article Title: Complex alterations in microglial M1/M2 markers during the development of epilepsy in two mouse models.

doi: 10.1111/epi.12960

Figure Lengend Snippet: Figure 3. Phasic increased hippocampal mRNA expression of M1 and M2 markers in i.h. kainate post-SE mice over disease progression reveal early and late marker expression. (A) M1-associated TNFa, IL-1b, and CD11b were all increased in SE mice compared to No-SE controls at the acute and late chronically epileptic time points. No changes in M1 expression were observed at the early chronic time point (n = 6– 12 per group). Antigen presentation M1-associated marker CD16 was increased in SE animals only acutely post-SE. Similarly astrocytic activation marker GFAP was only significantly increased in SE mice at the acute time point, and did show a trend of increased expression in the late chronic epileptic period. (B) Selected M2 markers were increased in SE animals in the i.h. kainate model. No significant increases in any M2-associated marker were measured at either the acute or early chronic time points; however, oxidative stress regula- tor enzyme Arg1, mannose receptor CD206, and anti-inflammatory cytokine IL-10 were increased in SE mice in the late chronic time point in this model. No significant changes to Ym1 or IL-4 expression were observed at this same time point. Mean SEM were shown for each group. n = 5–12 per group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Two-way ANOVA with Bonferroni post- test used for statistical analysis of all data sets. Epilepsia ILAE

Article Snippet: Intracellular Arg1 (Fluorescein, IC5868F; R&D Systems, Minneapolis, MN, U.S.A.) and Ym1 (nonconjugated, MAB2446; R&D Systems; secondary – PE conjugated) were visualized after cell permeabilization.

Techniques: Expressing, Biomarker Discovery, Marker, Immunopeptidomics, Activation Assay

Journal: Journal of Neuroinflammation

Article Title: Chi3l3: a potential key orchestrator of eosinophil recruitment in meningitis induced by Angiostrongylus cantonensis

doi: 10.1186/s12974-018-1071-2

Figure Lengend Snippet: Elevation of Chi3l3 expression in the brain is an important characteristic in non-permissive host mice but not in permissive host rats during AC infection. a Expression profile of significant TCGs after AC infection. The horizontal axis represents genes, and vertical coordinates represents time points. D0 is the normal control group. D2, D7, D14, and D21 represent 2, 7, 14, and 21 dpi, respectively. b Expression patterns of TCGs. The x -axis represents time (with the unit as day), and the y -axis represents the normalized gene expression levels. c The mRNA levels of Chi3l3 in brains of mice infected with AC at 0, 1, 3, 7, 14, and 21 dpi. Mouse β-actin mRNA was used as an internal control. * P < 0.05. infected groups vs control group; ## P < 0.01, 14 dpi group vs 21 dpi group. d The mRNA levels of Chi3l3 in brains of rats infected with AC at 0, 1, 3, 7, 14, and 21 dpi. Rat B2m and Hprt1 mRNA levels were used as internal controls. * P < 0.05, ** P < 0.01, infected groups vs control group. e The protein levels of Chi3l3 in brains of mice infected with AC at 0, 7, 14, 21, and 28 dpi. β-Actin was used as an internal control. * P < 0.05, ** P < 0.01, *** P < 0.001, infected groups vs uninfected groups in the corresponding part. f A gene set enrichment analysis of transcriptome data was performed by comparing the mRNA levels of CLPs and chitinases in brains of mice infected with AC at 0, 2, 7, 14, and 21 dpi. Data information: In c – e , data are presented as mean ± SD. (Student’s t test)

Article Snippet: The following reagents were used in this study: Recombinant Murine M-CSF (315-02; Peprotech), Recombinant Mouse Chi3l3 (2446-CH-050, R&D Systems), and Recombinant Mouse IL-13 (413-ML-025/CF, R&D Systems).

Techniques: Expressing, Infection, Control, Gene Expression

Journal: Journal of Neuroinflammation

Article Title: Chi3l3: a potential key orchestrator of eosinophil recruitment in meningitis induced by Angiostrongylus cantonensis

doi: 10.1186/s12974-018-1071-2

Figure Lengend Snippet: Eosinophil percentage coordinates with Chi3l3 derived from inflammatory macrophages in brains of AC-infected mice. a Chi3l3 + cells were observed in a gate of SSC and Chi3l3 + (A), within the gate (A). CD45 hi F4/80 + and F4/80 + CD11b + populations are shown in (C) and (D), respectively. b Chi3l3 + cells were characterized by CCR2 + gate (C) or CX3CR1 + gate (F) separately. c (A–D) The co-localization of Chi3l3 or Iba1 (A), CD11b or Chi3l3 (B), and DAPI (C) in cerebrum of AC-infected mice. (D) is the merge of (A), (B), and (C). (E–H) Chi3l3 or Iba1 (E), CD11b or Chi3l3 (F), and DAPI (G) in the cerebellum of AC-infected mice. (H) is the merge of (E), (F), and (G). Scale bars indicate 20 μm. Scale bar in (I) indicates 5 μm. d The percentages of Chi3l3 + CD45 lo F4/80 + cells in BMNCs of mice at 7, 14, and 21 dpi are presented. *** P < 0.001, **** P < 0.0001, AC-infected groups vs control group. Chi3l3 + CD45 hi F4/80 pos cells in BMNCs of mice at 7, 14, and 21 dpi are presented. #### P < 0.0001, AC-infected groups vs control group. $$ P < 0.01, $$$ P < 0.001, $$$$ P < 0.0001. e The percentages of eosinophils in BMNCs of mice infected with AC at 7, 14, and 21 dpi. There were 5–10 mice per group. Ctrl indicates the concurrent control. * P < 0.05, **** P < 0.0001, AC-infected groups vs control group. ### P < 0.001, AC-infected 14 dpi group vs AC-infected 7 dpi group. $$ P < 0.01, AC-infected 21 dpi group vs AC-infected 14 dpi group. f The percentages of eosinophils in peripheral blood mononuclear cells (PBMCs) of mice infected with AC at 7, 14, and 21 dpi; 5–10 mice per group. Ctrl indicates the concurrent control. * P < 0.05, AC-infected groups vs control group. g Clophosome and saline were injected into BALB/c mice intravenously 1 day before of AC infection, followed by five intravenous challenges every 4 days. At 22 dpi, the brain samples were collected. h Brain Chi3l3 mRNA levels ( g ). i Histopathological changes of the brain ( g ) (scale bar, 50 μm). Data information: In ( d-h ), data are presented as mean ± SD. (Student’s t test)

Article Snippet: The following reagents were used in this study: Recombinant Murine M-CSF (315-02; Peprotech), Recombinant Mouse Chi3l3 (2446-CH-050, R&D Systems), and Recombinant Mouse IL-13 (413-ML-025/CF, R&D Systems).

Techniques: Derivative Assay, Infection, Control, Saline, Injection

Journal: Journal of Neuroinflammation

Article Title: Chi3l3: a potential key orchestrator of eosinophil recruitment in meningitis induced by Angiostrongylus cantonensis

doi: 10.1186/s12974-018-1071-2

Figure Lengend Snippet: Functional protein association networks for the genes correlated with Chi3l3. a A full view of the protein association network for the genes highly correlated with Chi3l3. b The sub-network specifically centered on Chi3l3. c Functional enrichment for the genes in cluster 1 of gene expression patterns. The top 10 biological processes with the observed gene count as well as the false discovery rate are listed. d IL-13 mRNA levels in normal and AC-infected mouse brains. * P < 0.05, AC-infected 21 dpi group vs control group. e A mathematical model for Chi3l3-IL-13 positive feedback and bifurcation analysis. (A, B) Fitting the model to the experimental data. (C, D) Bistability of Chi3l3 and IL-13 with respect to the parameter value ( K 2 ). Data information: In ( d – e ), data are presented as mean ± SD. (Student’s t test)

Article Snippet: The following reagents were used in this study: Recombinant Murine M-CSF (315-02; Peprotech), Recombinant Mouse Chi3l3 (2446-CH-050, R&D Systems), and Recombinant Mouse IL-13 (413-ML-025/CF, R&D Systems).

Techniques: Functional Assay, Gene Expression, Infection, Control

Journal: Journal of Neuroinflammation

Article Title: Chi3l3: a potential key orchestrator of eosinophil recruitment in meningitis induced by Angiostrongylus cantonensis

doi: 10.1186/s12974-018-1071-2

Figure Lengend Snippet: A positive feedback loop between Chi3l3 from macrophages and IL-13 from T lymphocytes in vitro . a The percent of IL-5 + cells in CD3 + CD4 + spleen cells were detected by flow cytometry. b The percent of IL-13 + cells in CD3 + CD4 + cells. * P < 0.05, AC-infected D21 (21 dpi) group vs control group. c , d Spleen cells isolated from normal mice and AC-infected mice were stimulated with sAg (25 μg/mL) or Chi3l3 (10 ng/mL) for 72 h in vitro. A summary of the percentages of IL-13 + CD3 + CD4 + cells following stimulation with sAg and Chi3l3 in normal mice. * P < 0.05, Chi3l3 group vs control group. e Western blot analysis of Chi3l3, JMJD3, CREB1, CEBPB, KLF4, Y641 phospho-STAT6, STAT6, and PPARγ of BMDMs in the presence of sAg, IL-13, sAg+IL-13, and LPS for 24 h. f Cells were cultured for 24 h in medium alone or treated with sAg, IL-13, or sAg+IL-13, and then, the OCR was monitored using the Seahorse Bioscience extracellular flux analyzer in real time. Dotted lines indicate incubation of cells with the indicated compounds. g Basal OCR of BMDMs cultured for 24 h in medium alone or treated with sAg, IL-13, or sAg+IL-13. ** P < 0.01, *** P < 0.001, **** P < 0.0001, sAg group, IL-13 group, sAg+IL-13 group vs control group. # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001, sAg group and IL-13 group vs sAg+IL-13 group. h SRC of BMDMs cultured for 24 h in medium alone or treated with sAg, IL-13, or sAg+IL-13. ** P < 0.01, *** P < 0.001, **** P < 0.0001, sAg group, IL-13 group, sAg+IL-13 group vs control group. # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001, sAg group and IL-13 group vs sAg+IL-13 group. i Maximal OCR of BMDMs cultured for 24 h in medium alone or treated with sAg, IL-13, or sAg+IL-13. ** P < 0.01, *** P < 0.001, **** P < 0.0001, sAg group, IL-13 group, sAg+IL-13 group vs control group. # P < 0.05, ## P < 0.01, ### P < 0.001, #### P < 0.0001, sAg group and IL-13 group vs sAg+IL-13 group. Data information: In ( a – d, f – h ), data are presented as mean ± SD (Student’s t test)

Article Snippet: The following reagents were used in this study: Recombinant Murine M-CSF (315-02; Peprotech), Recombinant Mouse Chi3l3 (2446-CH-050, R&D Systems), and Recombinant Mouse IL-13 (413-ML-025/CF, R&D Systems).

Techniques: In Vitro, Flow Cytometry, Infection, Control, Isolation, Western Blot, Cell Culture, Incubation

Journal: Journal of Neuroinflammation

Article Title: Chi3l3: a potential key orchestrator of eosinophil recruitment in meningitis induced by Angiostrongylus cantonensis

doi: 10.1186/s12974-018-1071-2

Figure Lengend Snippet: Top 10 genes ranked according to the maximal changing rate of genes in cluster 1 of gene expression patterns

Article Snippet: The following reagents were used in this study: Recombinant Murine M-CSF (315-02; Peprotech), Recombinant Mouse Chi3l3 (2446-CH-050, R&D Systems), and Recombinant Mouse IL-13 (413-ML-025/CF, R&D Systems).

Techniques: Gene Expression, Expressing