Journal: International Immunology

Article Title: Integrin CD11b provides a new marker of pre-germinal center IgA + B cells in murine Peyer’s patches

doi: 10.1093/intimm/dxab113

Figure Lengend Snippet: CD11b + IgA + B cells are pre-GC B cells interacting with CD4 + T cells. (A) Heatmap indicating the log fold change (CD11b + IgA + B cells/CD11b − IgA + B cells) in expression of indicated genes by microarray analysis. (B) Expression levels of indicated GC specific genes and non-GC specific genes and a pre-GC gene were analyzed by qPCR and normalized to those from the β-actin gene. The mean of relative expression levels of CD11b − IgA + B cells is taken as 1. Each spot represents the mean of technical triplicates. Bar graphs show the mean values (±SD) of three or four independent measurements. Data were analyzed by ANOVA followed by Tukey’s multiple comparisons. (C) Flow cytometry histograms depicting expression of surface markers of CXCR4 and CD86 in CD11b + IgA + B cells and CD11b − IgA + B cells. Representative data are from at least five independent experiments. (D) Flow cytometry data of singlet IgA + B cells and conjugated CD4 + IgA + cells. (E) Flow cytometry data of CD11b − IgA + and CD11b + IgA + PP B cells with CD4 + T cells in the conjugated gate. (F) Percentage of the CD4 + T cells in conjugation with CD11b + IgA + and CD11b − IgA + PP B cells. Bar graphs show the mean values (±SD) of six independent measurements. Data were compared by two-tailed unpaired Student’s t test. (G) Imaging of sorted CD11b + IgA + PP B cells conjugating with CD4 + T cells. Scale bar, 20 μm.

Article Snippet: Microarray analysis was performed with a SurePrint G3 Mouse GE v2 8x60K Microarray Chip (Affymetrix) to detect the gene expression.

Techniques: Expressing, Microarray, Flow Cytometry, Conjugation Assay, Two Tailed Test, Imaging

Journal: Nature Communications

Article Title: Anoctamin 1 controls bone resorption by coupling Cl − channel activation with RANKL-RANK signaling transduction

doi: 10.1038/s41467-022-30625-9

Figure Lengend Snippet: a Microarray assays were performed in osteoclasts originated from Ano1 fl/fl ( n = 3) and Ctsk-Cre;Ano1 fl/fl ( n = 3) mice. The relative mRNA expression is depicted according to the color, red indicates upregulation and blue indicates downregulation. Shown are the mRNAs that changed more than 2 folds. b Pathway enrichment analysis (Gene Analytics) for genes expressed differentially between Ano1 fl/fl and Ctsk-Cre;Ano1 fl/fl osteoclasts. c Western blot analysis of NFATc1 protein level in Ano1 fl/fl and Ctsk-Cre;Ano1 fl/fl osteoclasts after treatment with or without RANKL mice (top). The quantification of NFATc1 protein level in osteoclasts (below). d Western blot analysis of NFATc1 protein level in WT and Ano1 TG osteoclasts after treatment with or without RANKL (top). The quantification of NFATc1 protein level in osteoclasts (below). e Immunofluorescence of Ano1 (green) and RANK (red) in osteoclasts treated with or without RANKL by confocal microscopy. Representative images are shown. Scale bar, 10 μm. f Coimmunoprecipitation of Ano1 and RANK in osteoclasts after treatment with or without RANKL. g Coimmunoprecipitation of Ano1 and RANK in RANKL-induced osteoclasts after treatment with Ano1 inhibitor Benzbromarone (10 μM). h Coimmunoprecipitation of Ano1 and RANK in RANKL-induced osteoclasts after overexpression of Ano1 or Ano1 with E702Q and E705Q mutants. i Coimmunoprecipitation of RANK and TRAF6 in RANKL-induced osteoclasts transfected with Ano1 siRNA or its negative control. j Western blot analysis of the phosphorylation levels of Syk, Btk, and Plcγ2 in osteoclasts (top). The quantification of the phosphorylation level of Syk, Btk, and Plcγ2 in osteoclasts (below). k Resting [Ca2+] i in osteoclasts from Ano1 fl/fl mice and Ctsk-Cre;Ano1 fl/fl mice, n = 40 ( Ano1 fl/fl ) and n = 55 ( Ctsk-Cre;Ano1 fl/fl ) cells pooled from three independent experiments. l Western blot analysis of p-CaMKIV, p-Creb, and c-Fos protein levels in osteoclasts (top). The quantification of p-CaMKIV, p-Creb, and c-Fos protein levels in osteoclasts (below). All data are the mean ± s.e.m. from three independent experiments. Two-tailed unpaired Student’s t -test was used for statistical evaluations of two group comparisons. Statistical analysis with more than two groups was performed with two-way analysis of variance (ANOVA) with Šídák post-hoc test to determine group differences. Source data are provided as a Source Data file.

Article Snippet: Agilent SurePrint G3 Mouse Gene Expression v2 8x60K Microarray (Design ID: 074809) chip was used in this project.

Techniques: Microarray, Expressing, Western Blot, Immunofluorescence, Confocal Microscopy, Over Expression, Transfection, Negative Control, Two Tailed Test

Journal: Journal of Experimental Botany

Article Title: A salt-regulated peptide derived from the CAP superfamily protein negatively regulates salt-stress tolerance in Arabidopsis

doi: 10.1093/jxb/erv263

Figure Lengend Snippet: Identification of AtCAPE1 in Arabidopsis . (A) Deduced amino acid sequence of the PROAtCAPE1 product. The predicted secretion signal peptide is underlined. The CAP domain is shaded in grey. The putative AtCAPE1 peptide is shaded in red. The cleavage site predicted to produce AtCAPE1 is indicated with an arrowhead. The putative cleavage signal motif is double-underlined. (B) LC-MS/MS spectrum of the synthetic AtCAPE1. The y-ion is the C-terminal fragments after peptide bond cleavage while the b-ion is the N-terminal fragments. (C) LC-MS/MS spectrum of the identified AtCAPE1 in Arabidopsis . (D) Schema representing the construct used for constitutive overexpression of enhanced yellow fluorescent protein (eYFP)-tagged PROAtCAPE1. The green box shows the CNYx motif. The putative CAPE is shown in red. The numbers indicate the predicted molecular weight of precursor protein tagged with eYFP (45.7kDa) and the cleaved precursor tagged with eYFP (26.3kDa). (E) Production of the precursor PROAtCAPE1 and the cleaved PROAtCAPE1 in CAPE1ox CNYD and CAPE1ox CNAD transgenic plants, where eYFP was fused to PROAtCAPE1 containing wild type (CNYD) and the mutated (CNAD) junction sequence, respectively. T3 seedlings derived from independent transgenic lines were sampled for western blotting with anti-GFP antibody. Coomassie blue staining was used for protein loading control. The lower panel shows the presence of the T-DNA insertions in the transgenic plants by genomic DNA PCR with the primer pair 35S-F’ and eYFP-R’ shown in (D).

Article Snippet: Agilent Arabidopsis (V4) Gene Expression Microarray 4×44k chips were used in this study.

Techniques: Sequencing, Liquid Chromatography with Mass Spectroscopy, Construct, Over Expression, Molecular Weight, Transgenic Assay, Derivative Assay, Western Blot, Staining

Journal: Nature Communications

Article Title: IL-21-mediated non-canonical pathway for IL-1β production in conventional dendritic cells

doi: 10.1038/ncomms8988

Figure Lengend Snippet: ( a ) Top panel: cDCs were rested 16 h and stimulated as indicated for 15 min. pSTAT3 was evaluated by flow cytometry. cDCs were gated as CD11c hi cells. Bottom panel: cDCs were rested 1 h, treated with 20 ng ml −1 IL-21, IL-6, IL-10 or Flt3L for 4 h, and intracellular pro-IL-1β analysed by flow cytometry. Shown are data representative of three experiments. ( b ) Summary of three experiments from lower panel of a . ** P values of the untreated sample compared with IL-21, IL-6 and IL-10 treated samples are 0.0002, 0.0017 and 0.0049, respectively; NS, P =0.4; error bars are means±s.e.m. ( c – e ) cDCs were stimulated with 100 ng ml −1 IL-21 or IL-10 for 1 h, then stimulated with 100 ng ml −1 LPS for 4 h, and the expression of Il1b ( c ), Il6 ( d ), and Tnf ( e ) mRNA were determined. Shown are combined results of 3 independent experiments; error bars are means±s.e.m. ( f ) Top panel: BMMs were rested without M-CSF for 16 h, treated with IL-21 or LPS for 4 h, and intracellular pro-IL-1β assessed by flow cytometry. Bottom panel: BMMs (gated as CD11c + F4/80 + cells) were rested and treated with IL-21 or LPS for 15 min. pSTAT3 was evaluated by flow cytometry. Data are representative of two experiments (total of 6 individual samples). ( g ) Top panel: CD4 + T cells were pre-activated with 5 μg ml −1 plate-bound anti-CD3+2 μg ml −1 soluble anti-CD28 for 3 days, washed, rested 16 h, treated with IL-21 for 4 h, and intracellular pro-IL-1β assessed by flow cytometry. Bottom panel: Rested CD4 + T cells were treated with IL-21 for 15 min. pSTAT3 was evaluated by flow cytometry. Data are representative of three experiments. Statistical analysis was performed by Student's t -test.

Article Snippet: Total mouse splenic CD4 + T cells were pre-activated with plate-bound anti-CD3+anti-CD28 for 3 days, washed and rested overnight, and not stimulated or stimulated with IL-21 for 4 h. Total RNA was isolated, and 5 μg per sample were used for mRNA purification using Dynal oligo(dT) beads (Invitrogen).

Techniques: Flow Cytometry, Expressing

Journal: Nature Communications

Article Title: IL-21-mediated non-canonical pathway for IL-1β production in conventional dendritic cells

doi: 10.1038/ncomms8988

Figure Lengend Snippet: ( a – g ), cDCs were rested 1 h, treated with IL-21 for 1 h; pre-activated CD4 + T cells were washed, rested 16 h, treated with IL-21 for 1 h, and ChIP-Seq performed for STAT3, H3K4me1, and H3K27ac. ( a ) Venn diagram showing overlapping and distinctive STAT3 binding sites in cDCs and CD4 + T cells. ( b – g ) For IL-21-induced-STAT3 binding sites that differentially exist in cDCs or CD4 + T cells, there were cell type-specific binding profiles of STAT3 ( b versus c ) and H3K4me1 ( d versus e ) and H3K27ac ( f versus g ) enhancer marks. Shown are normalized read densities near peak summits for cDC- or CD4 + T-cell specific STAT3 binding sites. ‘Dips' at the plot centres ( d – g ) represent open chromatin corresponding to nucleosome depletion. Data are representative of two experiments.

Article Snippet: Total mouse splenic CD4 + T cells were pre-activated with plate-bound anti-CD3+anti-CD28 for 3 days, washed and rested overnight, and not stimulated or stimulated with IL-21 for 4 h. Total RNA was isolated, and 5 μg per sample were used for mRNA purification using Dynal oligo(dT) beads (Invitrogen).

Techniques: ChIP-sequencing, Binding Assay

Journal: Nature Communications

Article Title: IL-21-mediated non-canonical pathway for IL-1β production in conventional dendritic cells

doi: 10.1038/ncomms8988

Figure Lengend Snippet: ( a ) Freshly isolated cDCs were rested 1 h, then stimulated with IL-21 for 4 h; CD4 + T cells were pre-activated for 3 days, then washed and rested 16 h, and stimulated with IL-21 for 4 h. Shown are genes differentially regulated by IL-21 in cDCs versus pre-activated CD4 + T cells. For cDCs, gene expression profiling was performed by microarray analysis, where cDCs were pooled from three independent experiments, as described in ref. . For pre-activated CD4 + T cells, gene expression profiling data were generated by RNA-Seq analysis. Shown are data from one of two similar experiments. ( b ) Il1b and Il21 expression in cDCs and CD4 + T cells not treated or stimulated with IL-21 as in a . Data are representative of 3 experiments. Error bars are technical duplicates of the representative experiment. ( c , d ) STAT3 binding, H3K4me1, H3K27ac, H3K4me3, and H3K27me3 marks at the Il1b locus in cDCs ( c ) and CD4 + T cells ( d ). Arrows in c indicate STAT3 binding sites at GAS1, GAS2 and GAS3 regions (GAS1: TTAgggGAA (−155 bp), TACcctGAA (−175 bp), TCCctgGAA (−195 bp); GAS2: TTTgggGAA (−2,452 bp), TTCctcCAA (−2,525 bp), TTCttcAAA (−2,549 bp); GAS3: TTGtgtGAA (−9,761 bp)). Arrows in d indicate the STAT3 binding sites identified in cDCs, but no STAT3 binding was seen at these sites in CD4 + T cells. ( e , f ) STAT3 binding, H3K4me1, H3K27ac, H3K4me3 and H3K27me3 marks at the Il21 gene locus in cDCs ( e ) and CD4 + T cells ( f ). Arrow in f indicates the STAT3 binding site at the GAS motif in the Il21 promoter region. Arrow in e indicates this same site, but no STAT3 binding was seen at this site in cDCs. Data are representative of two experiments. Statistical analysis was performed by Student's t -test.

Article Snippet: Total mouse splenic CD4 + T cells were pre-activated with plate-bound anti-CD3+anti-CD28 for 3 days, washed and rested overnight, and not stimulated or stimulated with IL-21 for 4 h. Total RNA was isolated, and 5 μg per sample were used for mRNA purification using Dynal oligo(dT) beads (Invitrogen).

Techniques: Isolation, Expressing, Microarray, Generated, RNA Sequencing Assay, Binding Assay

Journal: Nature Communications

Article Title: IL-21-mediated non-canonical pathway for IL-1β production in conventional dendritic cells

doi: 10.1038/ncomms8988

Figure Lengend Snippet: ( a ) cDCs were rested 1 h, treated with 100 ng ml −1 IL-21 or LPS at the indicated time points, and intracellular pro-IL-1β expression was determined. β-actin was used as control. Shown is one of two similar experiments. ( b ) cDCs were treated as in a , with 5 mM ATP added 1 h prior indicated time points in LPS-stimulated samples, and the secretion of IL-1β was determined by enzyme-linked immunosorbent assay (ELISA). Shown are combined results of two independent experiments; error bars are means±s.e.m. ( c ) CD4 + T cells from WT or Il1r −/− mice were cultured in Th17 cell differentiation conditions for 2 days, then supernatant from a 24 h, IL-21-treated cDC culture was added to the Th17 cells and incubated for 2 days, with or without addition of 10 μg ml −1 of anti-IL-1β. Expression of IL-2Rα (MFI) was determined by flow cytometry. The amount of biologically active IL-1β was determined using a standard curve constructed by assaying recombinant IL-1β. Shown are the combined results of two independent experiments with total of six samples. ( d , e ) WT, Casp1 −/− , Nlrp3 −/− and Pycard −/− cDCs were rested 1 h. In d , cDCs were then treated with 100 ng ml −1 LPS for 20–24 h with 5 mM ATP added in the final 1 h, and IL-1β assessed. Data are from two experiments; error bars are means±s.e.m. In e , cDCs were then treated with IL-21 for 20–24 h and IL-1β protein determined. Data are from five experiments. P values of IL-21-treated WT samples as compared with Casp1 −/− , Nlrp3 −/− and Pycard −/− samples are 0.99, 0.22 and 0.96, respectively; error bars are means±s.e.m. ( f ) cDCs from Ripk3 −/− , Ripk3 +/− Casp8 +/− and Ripk3 −/− Casp8 −/− mice were treated as in e , and IL-1β assessed. Data shown are from three experiments. P values of IL-21-treated Ripk3 −/− sample compared with Ripk3 +/− Casp8 +/− and Ripk3 −/− Casp8 −/− samples are 0.57 and 0.93, respectively. In b and d – f , IL-1β production in the culture supernatant was determined by ELISA. Pro-IL-1β induced by IL-21 in the culture supernatant was minimal, based on a pro-IL-1β-specific ELISA. Statistical analysis was performed by Student's t -test.

Article Snippet: Total mouse splenic CD4 + T cells were pre-activated with plate-bound anti-CD3+anti-CD28 for 3 days, washed and rested overnight, and not stimulated or stimulated with IL-21 for 4 h. Total RNA was isolated, and 5 μg per sample were used for mRNA purification using Dynal oligo(dT) beads (Invitrogen).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Cell Differentiation, Incubation, Flow Cytometry, Construct, Recombinant