Journal: Advanced Science

Article Title: OTUD6A in Airway Epithelial Cells Exacerbates Allergic Asthma by Promoting Airway Inflammation and Airway Remodeling Through Deubiquitination of hResistin/mRELMα

doi: 10.1002/advs.202516355

Figure Lengend Snippet: OTUD6A expression is upregulated in airway epithelial cells in asthma. A‐B) Immunohistochemical (IHC) staining and quantification of OTUD6A in human lung tissues from healthy controls ( n = 2) and asthmatic patients ( n = 2). Scale bars: 100 µm. C‐D) Western blot analysis of OTUD6A expression in lung tissue from HDM‐CAM (C) and HDM‐AAM (D) mice. E‐F) Representative images of IHC for OTUD6A on mouse lung sections of HDM‐CAM and corresponding quantitative analysis ( n = 5). Scale bars: 100 µm. G‐H) Representative images of IHC for OTUD6A on mouse lung sections of HDM‐AAM and corresponding quantitative analysis ( n = 5). Scale bars; 100 µm. I‐J) Immunofluorescence staining for OTUD6A in the mouse lung tissue of HDM‐AAM and corresponding quantitative analysis ( n = 5). Scale bars: 50 µm. K) Western blot analysis of OTUD6A protein levels in BEAS‐2B cells stimulated with HDM (100 µg/mL) at indicated time points ( n = 5). L‐M) BEAS‐2B cells transfected with OTUD6A or control vector for 24 h. L) Western blot analysis of ZO‐1 and Occludin. M) RT‐qPCR analysis of IL‐25 , IL‐33 , and TSLP mRNA levels ( n = 5). N‐O) HBEpiC cells transfected with OTUD6A or control vector for 24 h. N) Western blot analysis of ZO‐1 and Occludin. O) ELISA analysis of IL‐33, IL‐25, and TSLP levels in cell supernatant ( n = 5). P‐Q) HBEpiC cells transfected with siOTUD6A or control vector for 48 h and stimulated with HDM (100 µg/mL) for 6 h. P) Western blot analysis of OTUD6A, ZO‐1, and Occludin. Q) ELISA analysis of IL‐33, IL‐25, and TSLP levels in cell supernatant ( n = 5). Data are presented as mean ± SEM. P values determined by two‐tailed unpaired t‐test or one‐way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Human IL‐25 (Cat# E‐EL‐H1648)/IL‐33 (E‐EL‐H2402)/TSLP (Cat# E‐EL‐H1598)/Resistin (Cat# E‐EL‐H1213) ELISA kits, and mouse IL‐25 (Cat# E‐EL‐M0187)/IL‐33 (Cat# E‐EL‐M2642)/TSLP (Cat# E‐EL‐M0642) ELISA kits were purchased from Elabscience (Wuhan, China).

Techniques: Expressing, Immunohistochemical staining, Immunohistochemistry, Western Blot, Immunofluorescence, Staining, Transfection, Control, Plasmid Preparation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Advanced Science

Article Title: OTUD6A in Airway Epithelial Cells Exacerbates Allergic Asthma by Promoting Airway Inflammation and Airway Remodeling Through Deubiquitination of hResistin/mRELMα

doi: 10.1002/advs.202516355

Figure Lengend Snippet: OTUD6A knockout alleviates asthma in HDM‐induced acute asthma model. A) Schematic diagram depicting the procedure of HDM‐AAM. B) AHR assessed via acetylcholine challenge ( n = 5). C) Serum IgE levels measured by ELISA ( n = 5). D) Representative H&E and PAS staining of lung tissue. Scale bars: 100 µm. E) Quantification of bronchial epithelial thickness ( n = 5). F) BALF eosinophil counts determined by Wright‐Giemsa staining ( n = 5). G‐J) IL‐5 and IL‐13 levels in BALF (G, H) and lung homogenates (I, J) measured by ELISA ( n = 5). K‐N) RT‐qPCR analysis of Il5 , Il13 , Muc5ac , and Muc5b mRNA levels in lung tissue ( n = 5). O) ELISA analysis of IL‐25, IL‐33, and TSLP levels in BALF ( n = 5). P) Western blot and quantification of ZO‐1 and Occludin in lung tissues. Data are presented as mean ± SEM. P values determined by one‐way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Human IL‐25 (Cat# E‐EL‐H1648)/IL‐33 (E‐EL‐H2402)/TSLP (Cat# E‐EL‐H1598)/Resistin (Cat# E‐EL‐H1213) ELISA kits, and mouse IL‐25 (Cat# E‐EL‐M0187)/IL‐33 (Cat# E‐EL‐M2642)/TSLP (Cat# E‐EL‐M0642) ELISA kits were purchased from Elabscience (Wuhan, China).

Techniques: Knock-Out, Enzyme-linked Immunosorbent Assay, Staining, Quantitative RT-PCR, Western Blot

Journal: Advanced Science

Article Title: OTUD6A in Airway Epithelial Cells Exacerbates Allergic Asthma by Promoting Airway Inflammation and Airway Remodeling Through Deubiquitination of hResistin/mRELMα

doi: 10.1002/advs.202516355

Figure Lengend Snippet: OTUD6A knockout mitigates asthma in HDM‐induced chronic asthma model. A) Schematic diagram depicting the procedure of HDM‐CAM. B‐D) Rn, Ers, and Rrs assessed via methacholine challenge ( n = 5). E) Representative H&E staining of lung tissue. Scale bars: 100 µm. F) Serum IgE levels measured by ELISA ( n = 5). G‐H) Total cell counts (G) and protein concentration (H) in BALF ( n = 5). I‐L) IL‐5 and IL‐13 levels in BALF (I, J) and lung homogenates (K, L) measured by ELISA ( n = 5). M‐R) RT‐qPCR analysis of Il5 , Il13 , Muc5ac , Tslp , Il25 , and Il33 mRNA level in lung tissues ( n = 5). S‐U) ELISA analysis of IL‐25, IL‐33, and TSLP levels in BALF ( n = 5). Data are presented as mean ± SEM. P values determined by one‐way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Human IL‐25 (Cat# E‐EL‐H1648)/IL‐33 (E‐EL‐H2402)/TSLP (Cat# E‐EL‐H1598)/Resistin (Cat# E‐EL‐H1213) ELISA kits, and mouse IL‐25 (Cat# E‐EL‐M0187)/IL‐33 (Cat# E‐EL‐M2642)/TSLP (Cat# E‐EL‐M0642) ELISA kits were purchased from Elabscience (Wuhan, China).

Techniques: Knock-Out, Staining, Enzyme-linked Immunosorbent Assay, Protein Concentration, Quantitative RT-PCR

Journal: Advanced Science

Article Title: OTUD6A in Airway Epithelial Cells Exacerbates Allergic Asthma by Promoting Airway Inflammation and Airway Remodeling Through Deubiquitination of hResistin/mRELMα

doi: 10.1002/advs.202516355

Figure Lengend Snippet: hResistin/PI3K mediates OTUD6A‐induced EMT process and the expression of epithelial‐derived alarmins. A‐G) BEAS‐2B cells transfected with siResistin for 48 h and then transfected with Flag‐OTUD6A for 24 h. A) RT‐qPCR analysis of TSLP , IL‐25 , IL‐33 mRNA level in BEAS‐2B cells ( n = 5). B) ELISA analysis of TSLP, IL‐25, and IL‐33 levels in cell supernatant ( n = 5). C) RT‐qPCR analysis of TGFB1 , ACTA2 , COL1A1 mRNA level in BEAS‐2B cells ( n = 5). D‐E) Western blot analysis of OTUD6A, hResistin, EMT, and PI3K/AKT markers. F) Immunofluorescence staining of E‐cadherin and N‐cadherin. Scale bars: 50 µm. G) Cell wound healing assay. Scale bars: 50 µm. H‐K) BEAS‐2B cells were pretreated with LY294002 (30 µ m ) for 30 min and transfected with Flag‐OTUD6A for 24 h. H) Western blot analysis of hResistin and EMT markers. I‐K) ELISA analysis of IL‐33, IL‐25, and TSLP levels in cell supernatant ( n = 5). Data are presented as mean ± SEM. P values determined by one‐way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Human IL‐25 (Cat# E‐EL‐H1648)/IL‐33 (E‐EL‐H2402)/TSLP (Cat# E‐EL‐H1598)/Resistin (Cat# E‐EL‐H1213) ELISA kits, and mouse IL‐25 (Cat# E‐EL‐M0187)/IL‐33 (Cat# E‐EL‐M2642)/TSLP (Cat# E‐EL‐M0642) ELISA kits were purchased from Elabscience (Wuhan, China).

Techniques: Expressing, Derivative Assay, Transfection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Immunofluorescence, Staining, Wound Healing Assay

Journal: Advanced Science

Article Title: OTUD6A in Airway Epithelial Cells Exacerbates Allergic Asthma by Promoting Airway Inflammation and Airway Remodeling Through Deubiquitination of hResistin/mRELMα

doi: 10.1002/advs.202516355

Figure Lengend Snippet: Lung‐specific OTUD6A knockdown attenuates HDM‐induced asthma. A) Schematic diagram depicting the procedure of HDM‐induced chronic asthma model. B) Western blot analysis of OTUD6A and mRELMα in lung tissues. C) AHR assessed via acetylcholine challenge ( n = 5). D) H&E staining of lung tissues. Scale bars: 100 µm. E) Serum IgE levels measured by ELISA ( n = 5). F‐G) Total cell counts (F) and protein concentration (G) in BALF ( n = 5). H‐I) IL‐5 (H) and IL‐13 (I) levels in BALF measured by ELISA ( n = 5). J‐L) RT‐qPCR analysis of Il4, Il5 , and Muc5ac mRNA levels in lung tissues ( n = 5). M) ELISA analysis of IL‐25, IL‐33, and TSLP levels in BALF (n = 5). N) Serum TGF‐β1 levels measured by ELISA ( n = 5). O) Masson's trichrome staining of lung sections. Scale bars: 100 µm. P) RT‐qPCR analysis of Tgfb1 , Acta2 , and Col1a1 mRNA levels in lung tissues ( n = 5). Q) Western blot analysis of Vimentin, TGFβ1, α‐SMA, and Fibronectin in the lung tissues. R) Western blot analysis of PI3K/AKT in the lung tissues. S) Western blot analysis of EMT markers in the lung tissues. Data are presented as mean ± SEM. P values determined by one‐way ANOVA (* p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Human IL‐25 (Cat# E‐EL‐H1648)/IL‐33 (E‐EL‐H2402)/TSLP (Cat# E‐EL‐H1598)/Resistin (Cat# E‐EL‐H1213) ELISA kits, and mouse IL‐25 (Cat# E‐EL‐M0187)/IL‐33 (Cat# E‐EL‐M2642)/TSLP (Cat# E‐EL‐M0642) ELISA kits were purchased from Elabscience (Wuhan, China).

Techniques: Knockdown, Western Blot, Staining, Enzyme-linked Immunosorbent Assay, Protein Concentration, Quantitative RT-PCR

Journal: The Journal of investigative dermatology

Article Title: IL-17E (IL-25) Enhances Innate Immune Responses during Skin Inflammation.

doi: 10.1016/j.jid.2019.01.021

Figure Lengend Snippet: Figure 1. IL-17E promotes skin inflammation and favors the preferential recruitment of neutrophils in vivo. Balb/c mice were intradermally injected with recombinant mouse IL-17E or saline in the back for 2 consecutive days. Skin samples were harvested on day 3. (a) Representative hematoxylin and eosin staining of skin from mice treated with IL-17E or saline (n ¼ 8). Original magnification 10. Scale bar ¼ 50 mm. (b) Profiling of the cellular infiltrate in the skin of saline- (black bars) and IL-17Eeinjected (red bars) mice by flow cytometry. Cumulative data from three independent experiments (n ¼ 8) are presented as absolute counts per cm2. (c) Representative flow cytometry plots of the experiment shown in b. Full-thickness line gates depict cell populations modified by IL- 17E treatment. Pseudocolor scale indicates absolute counts, and numbers in the gates refer to frequency relative to CD45þ cells. (d) Immunofluorescence analysis of skin sections stained for DAPI (blue), Ki67 (green), and cytokeratin 10 (red). Original magnification 40. Scale bar ¼ 20 mm. One representative result is shown (n ¼ 5). White arrows depict Ki-67 positive cells (left panel). Quantitative analysis of Ki67þ cells in the basal layer (n ¼ 5, right panel). (e) Quantitative analysis of the epidermal thickness based on hematoxylin and eosin-stained sections (n ¼ 5). (f) Gene expression analysis was performed using NanoString (Seattle, WA) technology in the skin of mice injected with IL-17E or saline for 3 hours. Volcano plot (left panel) displays differentially expressed genes upon IL-17E treatment. The y-axis corresponds to the mean expression value of log10 P-value, and the x-axis displays the log2 fold change value. The red dots represent the differentially expressed transcripts (threshold > 2 fold change). Heatmap (right panel) of differentially expressed mRNAs in IL-17Eeinjected versus saline (control) group (n ¼ 5). Expression values are colored based on their z-score after normalization across treatments.

Article Snippet: Mouse antihuman IL-17E mAb (clone 182293) and polyclonal goat anti-human myeloperoxidase antibody were from R&D Systems, rabbit anti-human cytokeratin 10 was from BioSB (Santa Barbara, CA), and rat anti-mouse Ki67 mAb (clone 16A8) and rat anti-mouse Ly6G (clone 1A8) were from Biolegend.

Techniques: In Vivo, Injection, Recombinant, Saline, Staining, Cytometry, Gene Expression, Expressing, Control

Journal: The Journal of investigative dermatology

Article Title: IL-17E (IL-25) Enhances Innate Immune Responses during Skin Inflammation.

doi: 10.1016/j.jid.2019.01.021

Figure Lengend Snippet: Figure 3. IL-17E neutralization through blocking antibodies ameliorates imiquimod-induced skin inflammation. Wild-type balb/c mice were treated with imiquimod for 3 consecutive days on the shaved back skin and one ear. Next, 100 mg of neutralizing IL-17E antibody or isotype control were injected intraperitoneally1 day before the first application and 1 hour before every application. Skin and ear tissue samples were harvested on day 4. (a) Daily body weight variation and percentage of increase in ear swelling expressed as mean standard error of the mean (n ¼ 5). (b) Representative hematoxylin and eosin staining of the back skin and quantitative analysis of the epidermal thickness of imiquimod-treated mice injected with anti-IL-17E (n ¼ 11) or isotype control

Article Snippet: Mouse antihuman IL-17E mAb (clone 182293) and polyclonal goat anti-human myeloperoxidase antibody were from R&D Systems, rabbit anti-human cytokeratin 10 was from BioSB (Santa Barbara, CA), and rat anti-mouse Ki67 mAb (clone 16A8) and rat anti-mouse Ly6G (clone 1A8) were from Biolegend.

Techniques: Neutralization, Blocking Assay, Control, Injection, Staining

Journal: The Journal of investigative dermatology

Article Title: IL-17E (IL-25) Enhances Innate Immune Responses during Skin Inflammation.

doi: 10.1016/j.jid.2019.01.021

Figure Lengend Snippet: Figure 4. IL-17E neutralization affects innate immune cell infiltration during imiquimod-induced skin inflammation. Experimental condition, as in Figure 4. (a) Visualized t-SNE map of cells isolated from the skin of mice treated with imiquimod. Clusters (cell populations) were identified by manual gating. One representative result of each group is shown (n ¼ 10 for isotype control group; n ¼ 11 for a-IL-17E-treated group). (b) Based on clusters identified in a, median intensities for each marker were calculated and plotted as a heatmap. (c, d) Histogram showing the mean fluorescence intensity of the indicated markers in each cell cluster (c) belonging to the NK population (clusters 1e3) or (d) belonging to the neutrophil population (clusters 10 to 13) in the anti-IL-17E treated group. max, maximum; NK, natural killer; t-SNE, t-distributed stochastic neighbor embedding.

Article Snippet: Mouse antihuman IL-17E mAb (clone 182293) and polyclonal goat anti-human myeloperoxidase antibody were from R&D Systems, rabbit anti-human cytokeratin 10 was from BioSB (Santa Barbara, CA), and rat anti-mouse Ki67 mAb (clone 16A8) and rat anti-mouse Ly6G (clone 1A8) were from Biolegend.

Techniques: Neutralization, Isolation, Control, Marker

Journal: The Journal of investigative dermatology

Article Title: IL-17E (IL-25) Enhances Innate Immune Responses during Skin Inflammation.

doi: 10.1016/j.jid.2019.01.021

Figure Lengend Snippet: Figure 5. IL-17E promotes the recruitment of human neutrophils via activation of macrophages in a p38-dependent mechanism. (a) mRNA level of human IL-8 (left panel) and murine Cxcl1 and Cxcl2 (right panel) in IL-17Eetreated M2 macrophages from humans and mice, respectively. (b) IL-8 levels as assessed by ELISA in supernatants of primary keratinocytes (n ¼ 4) cultured in the presence of IL-17E or TNF (right panel). (c) mRNA levels assessed by quantitative PCR in M2 polarized macrophages pretreated for 30 minutes with a p38 MAPK (SB202190) or NF-kB inhibitor (JSH 23) and then stimulated with IL-17E for a further 6 hours. mRNA levels relative to the untreated condition are shown as mean standard error of the mean (n 5). (d) Immunoblotting analysis of M2 polarized macrophages stimulated with 100 ng/ml of IL-17E for the indicated time points (n ¼ 5, one representative result is shown). (e) Neutrophil migration induced by IL-8 (50 ng/ml) or IL-17E (100 ng/ml) in a classic Transwell assay (Nunc, Roskilde, Denmark) (left panel). M2 macrophages were left unstimulated (unstim.) or treated with IL-17E in the presence or not of SB202190 for 24 hours. Supernatants were retrieved and used to assess neutrophil migration. IL-8 neutralizing antibodies were added to the conditioned media of macrophages stimulated with IL-17E 30 minutes before assessing chemotaxis. Data from one representative experiment are expressed as relative to control media and presented as the mean standard error of the mean (n ¼ 3). (f) IL-8 protein levels assessed by ELISA in supernatants of M2 polarized macrophages. The protein levels relative to untreated condition are shown as mean standard error of the mean (n 5). Significant differences were assessed by Wilcoxon signed rank test. *P ¼ 0.01 to 0.05. Ctrl, control; min, minute; p-, phosphorylated.

Article Snippet: Mouse antihuman IL-17E mAb (clone 182293) and polyclonal goat anti-human myeloperoxidase antibody were from R&D Systems, rabbit anti-human cytokeratin 10 was from BioSB (Santa Barbara, CA), and rat anti-mouse Ki67 mAb (clone 16A8) and rat anti-mouse Ly6G (clone 1A8) were from Biolegend.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Real-time Polymerase Chain Reaction, Western Blot, Migration, Transwell Assay, Chemotaxis Assay, Control

Journal: The Journal of investigative dermatology

Article Title: IL-17E (IL-25) Enhances Innate Immune Responses during Skin Inflammation.

doi: 10.1016/j.jid.2019.01.021

Figure Lengend Snippet: Figure 6. An increase in IL-17ED cells may indicate a neutrophil infiltration in human skin inflammatory conditions. Expression of myeloperoxidase (green) in combination with IL-17E (red) and DAPI (blue) assessed by immunofluorescence in the skin of healthy volunteers (n ¼ 4) and patients with acute generalized exanthematous pustulosis (n ¼ 3) and pyoderma gangrenosum (n ¼ 4). Original magnification 20. Scale bar ¼ 50 mm. (b) Quantification of dermal IL-17Eþ and MPOþ cells in the samples, as in a. (c) Quantification of the expression of IL-17E in the epidermis of the samples shown in a. AGEP, acute generalized exanthematous pustulosis; MPO, myeloperoxidase; PG, pyoderma gangrenosum.

Article Snippet: Mouse antihuman IL-17E mAb (clone 182293) and polyclonal goat anti-human myeloperoxidase antibody were from R&D Systems, rabbit anti-human cytokeratin 10 was from BioSB (Santa Barbara, CA), and rat anti-mouse Ki67 mAb (clone 16A8) and rat anti-mouse Ly6G (clone 1A8) were from Biolegend.

Techniques: Expressing

Journal: Nature

Article Title: Thymic tuft cells promote an IL4-enriched medulla and shape thymocyte development

doi: 10.1038/s41586-018-0345-2

Figure Lengend Snippet: a, Co-localization of DCLK1 and RFP (IL25) in Flare25 thymus. Confocal maximal projection. Scale, 5 μm. n = 3 mice, 2 independent experiments. b, Differential expression of 20 tuft markers comparing sorted enterocytes and SI tuft cells (n = 3 mice) and thymic tuft cells (n = 4 mice) from Flare25 mice. Log2 fold change relative to mean expression. c, Expression (normalized reads from b ) of MHCII genes in SI and thymic tuft cells (mean +/− SD). * FDR < 0.00001. Lamp , FDR = 1. d, MHCII (I-A b ) surface staining on SI and thymic tuft cells. n = 3 mice; 2 independent experiments. e, Heatmap of select Tas2r genes comparing expression (normalized reads from b ) between thymic and SI tuft cells. f, Single cell RNA sequencing of RFP + (IL25 + ) thymic tuft cells. Heatmap shows selected tuft and Tas2r gene expression. Columns are single cells (n = 195), values are mean-centered log-normalized.

Article Snippet: A polyclonal goat anti-mouse IL25 antibody (R&D Systems, af1399) was used as a positive control.

Techniques: Quantitative Proteomics, Expressing, Staining, RNA Sequencing, Gene Expression

Journal: Nature

Article Title: Thymic tuft cells promote an IL4-enriched medulla and shape thymocyte development

doi: 10.1038/s41586-018-0345-2

Figure Lengend Snippet: a, DCLK1 bright mTEC counts. n = 5 mice per genotype; 3 independent experiments. b, mTECs from iALT.Flare25 double-reporter mice after 10-day tamoxifen treatment. Right, MHCII (I-A b ) surface expression within each gated population. n = 5 mice; 2 independent experiments. c, Single cell RNA sequencing of RFP + (IL25) Aire −/− thymic tuft cells. Heatmap shows selected tuft and Tas2r gene expression. Columns are single cells (n = 95), values are mean-centered log-normalized. d, PCA of single cell RNA sequencing data from RFP + tuft cells. Right, Gnat3 expression overlaid. e, DCLK1 staining with signal converted to Imaris surfaces; volumes > 5x mean single-cell volume (1×10 4 μm 3 ) are pseudocolored according to size. Right, pooled data. n = 3 mice per genotype. f, KRT10 surface area in thymic medulla (per unit area). Three regions analyzed per thymus. n = 4 mice per genotype; 2 independent experiments. g, h, Analysis of DCLK1 bright cells in Hipk2 fl/fl controls or FoxN1-Cre.Hipk2 fl/fl thymus. g, Confocal imaging of IF staining. h, Frequencies of DCLK1 hi MHCII lo or DCLK1 + MHCII hi mTECs from flow analysis. n = 6 mice; 3 independent experiments. e, g, Scale, 100 μm. a, e, f, h, Mean +/− SD; unpaired, parametric, 2-tailed Student’s t test.

Article Snippet: A polyclonal goat anti-mouse IL25 antibody (R&D Systems, af1399) was used as a positive control.

Techniques: Expressing, RNA Sequencing, Gene Expression, Staining, Imaging

Journal: Nature

Article Title: Thymic tuft cells promote an IL4-enriched medulla and shape thymocyte development

doi: 10.1038/s41586-018-0345-2

Figure Lengend Snippet: a, DT-treated Balb/cByJ. KN2 x C57BL/6. Aire-DTR F1 thymus (n = 10 mice) or non-Tg controls (n = 22 mice) gated on TCRβ int CD1d + iNKTs. Right, counts of IL4-producing (hCD2 + ) NKT2s (PLZF + ). b, DCLK1 intracellular staining in mTECs. n = 5 mice; 3 independent experiments. c, Thymic NKT2 (TCRβ int CD1d + PLZF + RORγt − ) cell counts in B6 (n = 15 mice), Pou2f3 −/− (n = 19 mice) and Trpm5 −/− (n = 10 mice). d, Thymic EOMES + TCRβ + CD8 + SP cell counts in B6 (n = 10 mice), Pou2f3 −/− (n = 11 mice) and Trpm5 −/− (n = 9 mice). e, Splenic total iNKT and NKT2 cell counts in B6 (n = 5 mice) and Pou2f3 −/− (n = 4 mice). f, Serum anti-IL25 autoantibody indices (AI) in nude mice transplanted with C57BL/6 (n = 3 mice) or Pou2f3 −/− (n = 12 mice) neonatal thymus and immunized with IL25 protein. IMM = immunized. Dashed line = average of C57BL/6 AI values plus 3 standard deiviations. of a, c–f, Mean +/− SD. a, e, Unpaired, parametric, 2-tailed Student’s t test. c, d, One-way, non-parametic ANOVA (Kruskal-Wallis test). a , c , d, Pooled from 3 independent experiments.

Article Snippet: A polyclonal goat anti-mouse IL25 antibody (R&D Systems, af1399) was used as a positive control.

Techniques: Staining

Journal: Nature

Article Title: Thymic tuft cells promote an IL4-enriched medulla and shape thymocyte development

doi: 10.1038/s41586-018-0345-2

Figure Lengend Snippet: a, DCLK1 and RFP (IL25) in C57BL/6 control and Flare25 thymus. Confocal maximal projection. Scale, 5 μm. n = 3 mice, 2 independent experiments. b, RFP (IL25) in C57BL/6 control and Flare25 mTECs (CD11c − CD45 − EPCAM + ). n = 5 mice; 3 independent experiments. c, Gating strategy for FACS sorting of CD11c − CD45 − Epcam + Ly51 − mTECs from Flare25 reporter mice for qPCR analysis of mTEC hi (RFP − MHCII hi ), mTEC lo (RFP + MHCII lo ), and thymic tuft (RFP + ) populations. d, qPCR analysis of expression of indicated genes of interest on populations sorted in ( c ) normalized to mTEC hi (mean +/− SD). n = 3 mice. Two-way non-parametric ANOVA with multiple comparisons. e, Representative confocal maximum projection (10 μm) stained for KRT8/KRT18 (red) and DCLK1 (green). Scale bars, 50 μm. n = 3 mice, 2 independent experiments. f–i, Expression levels (normalized reads from ) from bulk RNA sequencing of SI (n = 3 mice) and thymic tuft (n = 4 mice) cells. f, Major MHCI genes and B2m. FDR > 0.1. g, Major MHCII genes and CD74 in SI tuft cells. h, Minor MHCII genes in thymic tuft cells. i, Tas2r family members in thymic tuft cells. g–i, Mean +/− SD. g, i, Red line corresponds to a cutoff of 5 reads/million; * mean and SD fall above this cutoff.

Article Snippet: A polyclonal goat anti-mouse IL25 antibody (R&D Systems, af1399) was used as a positive control.

Techniques: Control, Expressing, Staining, RNA Sequencing