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

Article Title: IL-4 and IL-13 guide early thymic progenitors to mature towards dendritic cells

doi: 10.4049/jimmunol.1701186

Figure Lengend Snippet: (A) Thymic cells from HR+/+ IL-13Rα1-GFP reporter mice were depleted of Lin+ cells and the Lin-CD4-CD8- cells were stained with antibodies to CD25, CD44, c-Kit, and intracellular pSTAT6(Y641), pSTAT1(Y701), pSTAT1(S727), pSTAT3 (S727) and pSTAT3 (Y705). The CD25-CD44+c-Kit+GFP+ (HR+ETPs) and CD25-CD44+c-Kit+GFP- (HR-/PETPs) were then analysed ex vivo for phosphorylation of STAT6, STAT1, and STAT3 at the indicated aa residues in comparison to isotype control for the corresponding anti-STAT antibody. The histograms show a representative experiment and the bar graphs show the mean ± SD MFI results compiled from 3 independent experiments. **p<0.01 as determined by two-tailed, unpaired Student’s t-test. (B, C) Thymic cells from HR−/− mice were depleted of Lin+ cells and the Lin-CD4-CD8- cells were stained with antibodies to CD25, CD44 and c-Kit, sorted as CD25-CD44+c-Kit+HR−/− ETPs, and transduced with HR-RV to drive HR expression. The cells were stained with anti-Thy1.1 and anti-IL-13Rα1 (marker for HR expression) and sorted as IL-13Rα1+Thy1.1+ cells. HR-RV transduced ETPs were cultured in the presence of 10ng/ml IL-4 alone (IL-4) or with STAT1 (IL-4 +STAT1 I) or STAT6 (IL-4 + STAT6 I) inhibitor. Similar culture conditions were used with 20 ng/ml IL-13 alone (IL-13) or with STAT1 (IL-13 + STAT1 I) or STAT6 (IL-13 + STAT6 I) inhibitor. Culture without cytokine or inhibitor (NIL) was included for control purposes. (B) STAT1 and ( C) STAT6 phosphorylation was analyzed after 1 hour incubation by flow cytometry. The histograms show representative experiments for STAT1 and STAT6 phosphorylation while the bar graphs show the mean ± SD MFI results compiled from three independent experiments. *p<0.05, **p<0.01 as determined by one-way ANOVA.

Article Snippet: Anti-CD3 (145–2C11), anti-CD4 (RM4–5), anti-CD8 (53–6.7), anti-CD25 (7D4), anti-CD44 (IM7), anti-CD45 (30-F11), anti-Pax5 (1H9), anti-CD45.1 (A20), anti-CD11b (M1/70), anti-CD11c (HL3), anti-CD117 (2B8), anti-IFNγR1 (XMG1.2), anti-pSTAT6 Y641 (J71–773.58.11), anti-pSTAT3 S727 (49/p-Stat3), anti-pSTAT3 Y705 (4/P-STAT3), anti-Zbtb46 (U4–1374), anti-SIRPα (P84), and anti-CD90.1 (OX-7), were purchased from BD biosciences (San Jose, CA).

Techniques: Staining, Ex Vivo, Two Tailed Test, Transduction, Expressing, Marker, Cell Culture, Incubation, Flow Cytometry

Journal: iScience

Article Title: Sleep deprivation-induced sympathetic activation promotes pro-tumoral macrophage phenotype via the ADRB2/KLF4 pathway to facilitate NSCLC metastasis

doi: 10.1016/j.isci.2025.112321

Figure Lengend Snippet:

Article Snippet: anti- pSTAT6 , ImmunoWay , Cat# YP0256.

Techniques: Recombinant, Transfection, Enzyme-linked Immunosorbent Assay, Phagocytosis Assay, Sequencing, Software

Journal:

Article Title:

doi:

Figure Lengend Snippet: (A) Representative image demonstrates the peri-infarct area defined by Iba1 staining. Two randomly selected microscopic fields in the cortex (CTX) and 2 in the striatum (STR) of each section were subjected for analysis. Scale bar: 200 μm. (B) Representative images of coronal brain sections showing the phosphorylation of STAT6 (green) in Iba1+ microglia/macrophages (red) at indicated time points after tMCAO. Scale bar: 20 μm. (C) Representative images of coronal brain slices collected 3d after tMCAO showing the nuclear (DAPI, blue) localization of pSTAT6 (green) in Iba1+ (red) microglia/macrophages. Scale bar: 20 μm. (D) Quantification of the number of pSTAT6+Iba1+ cells in the ischemic regions indicated in B at different time points after tMCAO. n = 3–4 mice per group. **P ≤ 0.01, ***P ≤ 0.001 vs. 1d in CTX. ##P ≤ 0.01 vs. 1d in STR, 1-way ANOVA. (E and F) Flow cytometric analysis of pSTAT6 in brain cells 3d after tMCAO. (E) Representative dot plots demonstrate the gating strategy for microglia/macrophages (CD45+CD11b+), astrocytes (CD45–GLAST+), oligodendrocytes (CD45–O4+), and neurons (CD45–NeuN+). (F) Mean fluorescence intensity (MFI) of pSTAT6 in brain cells. n = 3 mice. ***P ≤ 0.01, Student’s t test. (G) Representative plot of STAT6 activation (pSTAT6) in microglia (CD45intermediate; CD45int) and macrophages (CD45hi) in pSTAT6+CD11b+ population. (H) STAT6 activation evaluation in microglia and macrophages by MFI of pSTAT6. n = 3 mice. **P ≤ 0.01, Student’s t test.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-pSTAT6 (phospho Y641) (Ab28829, Abcam, 1:100, used for both mouse and human samples); goat polyclonal anti-Iba1 (Ab5076, Abcam, 1:500, used for both mouse and human samples); rabbit polyclonal anti-Iba1 (019-19741, Wako, 1:500); mouse anti-NeuN (MAB377, MilliporeSigma,1:500); rabbit anti-NeuN (ABN78, MilliporeSigma,1:500); rat anti-CD16/CD32 (553142, BD Biosciences, 1:200); goat anti-CD206 (AF2535, R&D Systems,1:500); and goat polyclonal anti-Arg1 (sc-18351, Santa Cruz Biotechnology Inc., 1:200).

Techniques: Staining, Fluorescence, Activation Assay

Journal:

Article Title:

doi:

Figure Lengend Snippet: Brains were collected from WT and STAT6-KO mice 3d and 7d after 60 minutes tMCAO. (A–D) Analysis of microglia/macrophage phenotypes. (A) Representative images of microglia/macrophage marker Iba1 (red) and proinflammatory phenotype marker CD16 (green) double-staining. Scale bar: 50 μm. (B) Representative images of microglia/macrophage marker Iba1 (red) and antiinflammatory phenotype marker CD206 (green) double-staining. Scale bar: 50 μm. (C) Quantification of Iba1+CD16+ proinflammatory microglia/macrophages in ischemic areas at 3d and 7d after tMCAO. (D) Quantification of Iba1+CD206+ antiinflammatory microglia/macrophages in ischemic areas at 3d and 7d after tMCAO. n = 3–6 mice per group. **P ≤ 0.01, ***P ≤ 0.001 STAT6-KO vs. WT, Student’s t test. (E) The expression of CD16 and CD206 in pSTAT6+ and pSTAT6– microglia/macrophages (CD45+CD11b+) in ipsilateral hemisphere from WT mice was measured by flow cytometry 3d after tMCAO. Mean fluorescent intensity (MFI) of CD16 or CD206 in pSTAT6–CD45+CD11b+ (gray) and pSTAT6+CD45+CD11b+ (pink) populations was quantified. n = 5 per group. ***P ≤ 0.001 pSTAT6+ vs. pSTAT6– population, Student’s t test. (F) mRNA expression of proinflammatory and antiinflammatory markers were measured by RT-qPCR in the ipsilateral hemisphere 3d and 7d after ischemic stroke. n = 6 mice per group for 3d. n = 3 mice per group for 7d. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 STAT6-KO vs. WT, Student’s t test.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-pSTAT6 (phospho Y641) (Ab28829, Abcam, 1:100, used for both mouse and human samples); goat polyclonal anti-Iba1 (Ab5076, Abcam, 1:500, used for both mouse and human samples); rabbit polyclonal anti-Iba1 (019-19741, Wako, 1:500); mouse anti-NeuN (MAB377, MilliporeSigma,1:500); rabbit anti-NeuN (ABN78, MilliporeSigma,1:500); rat anti-CD16/CD32 (553142, BD Biosciences, 1:200); goat anti-CD206 (AF2535, R&D Systems,1:500); and goat polyclonal anti-Arg1 (sc-18351, Santa Cruz Biotechnology Inc., 1:200).

Techniques: Marker, Double Staining, Expressing, Flow Cytometry, Quantitative RT-PCR

Journal:

Article Title:

doi:

Figure Lengend Snippet: (A) Schematic illustration for the construction of WT/CX3CR1-GFP chimeric mice. (B–D) ImageStream analysis of pSTAT6 and Arg1 in CD45+CD11b+GFP– cells and CD45+CD11b+GFP+ macrophages in ipsilateral hemisphere of chimeric mice was performed 3d after tMCAO. (B) Representative images indicating pSTAT6 and Arg1 signal in both CD45+CD11b+GFP– cells and CD45+CD11b+GFP+ macrophages in ipsilateral brains. Scale bar: 7 μm. (C) Percentages of GFP– cells and GFP+ macrophages among CD45+CD11b+ cells were assessed in WT (left) and WT/CX3CR1-GFP chimeric mice (right). (D) Percentages of pSTAT6+ cells (left) and Arg1+ cells (right) observed among CD45+CD11b+GFP– cells and CD45+CD11b+GFP+ macrophages. n = 3 mice. (E) Chimeric mice were constructed by transplanting WT BM to WT recipients (WT/WT), STAT6-KO BM to WT recipients (WT/KO), or WT BM to STAT6-KO recipients (KO/WT) after irradiation of recipients. Chimeric mice were subjected to 60 minutes of tMCAO at 6 weeks after irradiation. (F and G) Infarct volume at 3d after tMCAO was quantified in MAP2-stained (green) coronal sections. Dashed lines outline the infarct area. n = 9–10 mice per group. Scale bar: 1 mm. (H and I) Neuronal death in chimeric mice was quantified by TUNEL (red) and NeuN (green) colabeling in infarct areas 3d after tMCAO. Scale bar: 10 μm. n = 3–5 mice per group. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 vs. WT/WT, 1-way ANOVA.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-pSTAT6 (phospho Y641) (Ab28829, Abcam, 1:100, used for both mouse and human samples); goat polyclonal anti-Iba1 (Ab5076, Abcam, 1:500, used for both mouse and human samples); rabbit polyclonal anti-Iba1 (019-19741, Wako, 1:500); mouse anti-NeuN (MAB377, MilliporeSigma,1:500); rabbit anti-NeuN (ABN78, MilliporeSigma,1:500); rat anti-CD16/CD32 (553142, BD Biosciences, 1:200); goat anti-CD206 (AF2535, R&D Systems,1:500); and goat polyclonal anti-Arg1 (sc-18351, Santa Cruz Biotechnology Inc., 1:200).

Techniques: Construct, Irradiation, Staining, TUNEL Assay

Journal:

Article Title:

doi:

Figure Lengend Snippet: (A) Immunostaining of Arginase 1 (Arg1, green) in Iba1+ microglia/macrophages (red) in the ischemic striatum (STR) and cortex (CTX) 3d after cerebral ischemia. Scale bar: 20 μm. (B) The expression of Arg1 in pSTAT6+ (pink) and pSTAT6– (gray) microglia/macrophage (CD45+CD11b+ gated) was measured 3d after tMCAO in ipsilateral hemisphere of WT mice by flow cytometry. Mean fluorescent intensity (MFI) of Arg1 was quantified. n = 5 mice. ***P ≤ 0.001 vs. pSTAT6– population, Student’s t test. (C) RT-qPCR analysis of Arg1 mRNA expression in WT and STAT6-KO microglia or macrophages without stimulation and after activating STAT6 signaling with IL-4 (20 ng/mL for 48 hours). Data were collected from 4 independent experiments. **P ≤ 0.01, *** P ≤ 0.001, 1-way ANOVA. (D) Western blot analysis of Arg1 protein expression in microglia or macrophages without stimulation and after activating STAT6 signaling with IL-4. Data were collected from 3 independent experiments. ***P ≤ 0.001, 1-way ANOVA.

Article Snippet: The following primary antibodies were used: rabbit polyclonal anti-pSTAT6 (phospho Y641) (Ab28829, Abcam, 1:100, used for both mouse and human samples); goat polyclonal anti-Iba1 (Ab5076, Abcam, 1:500, used for both mouse and human samples); rabbit polyclonal anti-Iba1 (019-19741, Wako, 1:500); mouse anti-NeuN (MAB377, MilliporeSigma,1:500); rabbit anti-NeuN (ABN78, MilliporeSigma,1:500); rat anti-CD16/CD32 (553142, BD Biosciences, 1:200); goat anti-CD206 (AF2535, R&D Systems,1:500); and goat polyclonal anti-Arg1 (sc-18351, Santa Cruz Biotechnology Inc., 1:200).

Techniques: Immunostaining, Expressing, Flow Cytometry, Quantitative RT-PCR, Western Blot

Journal: Cell reports

Article Title: Mass Cytometry Reveals Global Immune Remodeling with Multi-lineage Hypersensitivity to Type I Interferon in Down Syndrome

doi: 10.1016/j.celrep.2019.10.038

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit monoclonal anti-human phospho-STAT6 (Tyr641) (clone 18) , Fluidigm , Cat#3168012A; RRID:AB_2811103.

Techniques: Recombinant, Mass Cytometry, Software