Journal: Nature

Article Title: Gut-licensed IFNγ + NK cells drive LAMP1 + TRAIL + anti-inflammatory astrocytes

doi: 10.1038/s41586-020-03116-4

Figure Lengend Snippet: a, Analysis of CNS samples from naive Aldh1l1EGFP mice (n = 32). b, Top 42 surface markers and negative controls (neg. ctrls) expressed by astrocytes (Aldh1l1+) versus non-astrocytes (Aldh1l1−). c, Histograms depicting staining of surface-molecule antibodies (blue) and isotype control (red) gated on astrocytes (Aldh1l1+). d, Fluorescence-activated cell sorting (FACS) analysis of tdTomato+CX3CR1− astrocytes from the spinal cord of naive and EAE peak or EAE recovery Aldh1l1creERT2tdTomato mice (n = 7 for the EAE recovery group (except n = 6 for CD29); n = 6 mice otherwise). e, FACS plots of LAMP1+ astrocytes during EAE. f, g, LAMP1 expression in astrocytes from the spinal cord, measured as mean fluorescence intensity (MFI) (f) and as a percentage (g) from the screen in d (n = 6 mice for the naive group in f and the EAE peak group in g; n = 7 mice otherwise). Unpaired two-tailed t-test. Data are mean ± s.e.m. (f, g).

Article Snippet: For analysis of astrocytes derived from Aldh1l1 creERT2tdTomato reporter mice, CNS cells were stained with APC anti-mouse LAMP1 (BioLegend, 121614, 1:100) and PE–Cy7 anti-mouse TRAIL (BioLegend, 109311, 1:100) for 45 min on ice.

Techniques: Staining, Fluorescence, FACS, Expressing, Two Tailed Test

Journal: Nature

Article Title: Gut-licensed IFNγ + NK cells drive LAMP1 + TRAIL + anti-inflammatory astrocytes

doi: 10.1038/s41586-020-03116-4

Figure Lengend Snippet: a, EAE in Aldh1l1creERT2tdTomato mice. n = 6 naive, n = 7 peak, n = 7 recovery mice. b, Sorting schematic for tdTomato+ astrocytes. c, MFI of LAMP1 expression from screening. n = 6 naive, n = 7 peak, n = 7 recovery mice. Unpaired two-tailed t-test. Data are mean ± s.e.m.

Article Snippet: For analysis of astrocytes derived from Aldh1l1 creERT2tdTomato reporter mice, CNS cells were stained with APC anti-mouse LAMP1 (BioLegend, 121614, 1:100) and PE–Cy7 anti-mouse TRAIL (BioLegend, 109311, 1:100) for 45 min on ice.

Techniques: Expressing, Two Tailed Test

Journal: Nature

Article Title: Gut-licensed IFNγ + NK cells drive LAMP1 + TRAIL + anti-inflammatory astrocytes

doi: 10.1038/s41586-020-03116-4

Figure Lengend Snippet: a, Immunostaining of brain and spinal cord of mice transduced with Gfap-driven Cas9-2A-EGFP lentivirus. Representative images from n = 5 mice analysed. b, Percentage of GFP+GFAP+ cells by FACS. n = 5 mice. c, Quantification of astrocyte LAMP1 knockdown in astrocytes and microglia by FACS. n = 4 mice (sgScramble astrocyte); n = 5 mice otherwise. Unpaired two-tailed t-test. d, Gating strategy of CD4+ T cells from CNS. e, Analysis of splenic T cells from sgScramble- or sgLamp1-transduced mice. n = 5 mice sgLamp1 active-caspase 3; n = 6 mice otherwise. Unpaired two-tailed t-test. f, Analysis of CNS cells isolated from sgScramble- or sgLamp1-transduced mice. n = 6 per group. Unpaired two-tailed t-test. g, Analysis of CD11b+ cell apoptosis in sgScramble- or sgLamp1-transduced mice. n = 6 per group. Unpaired two-tailed t-test. h, Example gating used to sort LAMP1+ astrocytes from Aldh1l1creERT2tdTomato mice for scRNA-seq. i, Violin plots of genes, UMIs and cell-type markers for cells analysed by scRNA-seq from naive and EAE mice 17 days after induction. Astrocyte markers include Aqp4, Aldh1l1, Gfap, S100b, Aldoc, Slc1a3, Slc1a2 and Mfge8. j, Principal components used and gene expression scatterplots of LAMP1+ astrocytes isolated from Aldh1l1creERT2tdTomato mice. k, TRAIL expression on astrocytes from sgScramble- and sgLamp1-transduced mice 28 days after EAE induction. n = 5 sgScramble, n = 4 sgLamp1 mice. Unpaired two-tailed t-test. Data are mean ± s.e.m.

Article Snippet: For analysis of astrocytes derived from Aldh1l1 creERT2tdTomato reporter mice, CNS cells were stained with APC anti-mouse LAMP1 (BioLegend, 121614, 1:100) and PE–Cy7 anti-mouse TRAIL (BioLegend, 109311, 1:100) for 45 min on ice.

Techniques: Immunostaining, Transduction, Two Tailed Test, Isolation, Expressing

Journal: Nature

Article Title: Gut-licensed IFNγ + NK cells drive LAMP1 + TRAIL + anti-inflammatory astrocytes

doi: 10.1038/s41586-020-03116-4

Figure Lengend Snippet: a, Top, schematic of lentivirus vector. Bottom, EAE curves in mice treated with sgRNA against Lamp1 (sgLamp1) or control scramble sgRNA (sgScramble) (n = 19 sgScramble; n = 15 sgLamp1 mice). Experiment repeated three times. Two-way repeated measures analysis of variance (ANOVA). b, Quantification of immune cells in sgScramble- or sgLamp1-transduced mice (n = 9 sgScramble CD4+; n = 8 sgLamp1 CD4+; n = 6 mice otherwise). Two-tailed Mann–Whitney test. c, Quantification of the CD4+ T cell subset in sgScramble- or sgLamp1-transduced mice (n = 8 IFNγ+; n = 7 sgScramble IFNγ+IL-17+; n = 6 mice otherwise). Two-tailed Mann–Whitney test. d, Left, differential gene expression determined by bulk RNA-seq in sgScramble- or sgLamp1-transduced mice. Right, gene set enrichment analysis (GSEA) comparing sgScramble- and sgLamp1-transduced mice (day 19 after EAE induction; n = 3 mice per group). Gene Ontology (GO) terms are shown. FDR, false discovery rate; NES, normalized enrichment score. e, FACS analysis of activation of caspase-3 in CNS CD4+ T cells (n = 6 sgScramble; n = 5 sgLamp1 mice). Unpaired two-tailed t-test. f, Top, t-distributed stochastic neighbour embedding (t-SNE) plots of LAMP1+ astrocytes from naive or EAE Aldh1l1creERT2tdTomato mice (day 17 after EAE induction; n = 3 mice per group) analysed by scRNA-seq. Bottom, t-SNE plots of Lamp1 and Tnfsf10. g, Left, distribution of scRNA-seq clusters by condition. Right, number of cluster 0 (C0) cells (n = 812 cells). Data are mean ± s.e.m. (a–c, e).

Article Snippet: For analysis of astrocytes derived from Aldh1l1 creERT2tdTomato reporter mice, CNS cells were stained with APC anti-mouse LAMP1 (BioLegend, 121614, 1:100) and PE–Cy7 anti-mouse TRAIL (BioLegend, 109311, 1:100) for 45 min on ice.

Techniques: Plasmid Preparation, Two Tailed Test, MANN-WHITNEY, Expressing, RNA Sequencing Assay, Activation Assay

Journal: Nature

Article Title: Gut-licensed IFNγ + NK cells drive LAMP1 + TRAIL + anti-inflammatory astrocytes

doi: 10.1038/s41586-020-03116-4

Figure Lengend Snippet: a, EAE curves in sgScramble (n = 24) and sgTnfsf10 (n = 25) mice. Experiment repeated three times. Two-way repeated measures ANOVA. b, Quantification of T cell subsets (day 24 after EAE induction; n = 6 mice per group). One-tailed Mann–Whitney test. c, Activation of caspase-3 in CNS CD4+ T cells (n = 6 sgScramble; n = 5 sgTnfsf10 mice). Unpaired two-tailed t-test. d, Left, differential gene expression determined by bulk RNA-seq in astrocytes isolated from sgScramble (n = 4) or sgTnfsf10 (n = 3)-transduced mice (left) at 24 days after EAE induction. Right, GSEA comparing sgScramble- and sgTnfsf10-transduced mice. e, f, Left, t-SNE plots of LAMP1+TRAIL+ astrocytes from naive or EAE Aldh1l1creERT2tdTomato mice (day 17 after EAE induction; n = 3 mice per group) analysed by scRNA-seq. e, Right, number of cluster 0 cells (n = 1,089 cells). g, GSEA of C0 astrocytes. h, Top left, GO analysis of differentially upregulated pathways in C0 astrocytes. Reg., regulation. Bottom left, predicted upstream regulation of the C0 transcriptional signature using Ingenuity Pathway Analysis (Qiagen). Inc. expr., increased expression; dec. expr., decreased expression; pred. activ., predicted activation. Right, t-SNE plots of Ifngr1 and Irf1. Data are mean ± s.e.m. (a–c).

Article Snippet: For analysis of astrocytes derived from Aldh1l1 creERT2tdTomato reporter mice, CNS cells were stained with APC anti-mouse LAMP1 (BioLegend, 121614, 1:100) and PE–Cy7 anti-mouse TRAIL (BioLegend, 109311, 1:100) for 45 min on ice.

Techniques: One-tailed Test, MANN-WHITNEY, Activation Assay, Two Tailed Test, Expressing, RNA Sequencing Assay, Isolation

Journal: Nature

Article Title: Gut-licensed IFNγ + NK cells drive LAMP1 + TRAIL + anti-inflammatory astrocytes

doi: 10.1038/s41586-020-03116-4

Figure Lengend Snippet: a, Quantification of TRAIL knockdown using FACS in astrocytes and microglia. n = 5 mice per group. Unpaired two-tailed t-test. b, Analysis of CNS cell numbers in sgScramble- or sgTnfsf10-transduced mice. n = 6 mice per group. Unpaired two-tailed t-test. c, Control analyses of splenic T cells from knockdown mice. n = 6 mice per group. Unpaired two-tailed t-test. d, Analysis of CD11b+ cell apoptosis in sgScramble- or sgTnfsf10-transduced mice. n = 6 mice per group. Unpaired two-tailed t-test. e, Cytokine production and recall response of splenic T cells isolated from knockdown mice. n = 6 mice per group. n = 8 sgScramble CPM, n = 5 sgScramble IL-17 mice. Unpaired two-tailed t-test. f, Sorting schematic of LAMP1+TRAIL+ astrocytes from Aldh1l1creERT2tdTomato mice for scRNA-seq. g, Principal components used in scRNA-seq analysis of LAMP1+TRAIL+ astrocytes isolated from Aldh1l1creERT2tdTomato mice from naive and EAE mice at 17 days after induction. h, Violin plots of genes, UMIs and cell-type markers for cells analysed. Astrocyte markers include Aqp4, Aldh1l1, Gfap, S100b, Aldoc, Slc1a3, Slc1a2and Mfge8. Data are mean ± s.e.m.

Article Snippet: For analysis of astrocytes derived from Aldh1l1 creERT2tdTomato reporter mice, CNS cells were stained with APC anti-mouse LAMP1 (BioLegend, 121614, 1:100) and PE–Cy7 anti-mouse TRAIL (BioLegend, 109311, 1:100) for 45 min on ice.

Techniques: Two Tailed Test, Isolation

Journal: Nature

Article Title: Gut-licensed IFNγ + NK cells drive LAMP1 + TRAIL + anti-inflammatory astrocytes

doi: 10.1038/s41586-020-03116-4

Figure Lengend Snippet: a, Quantitative PCR (qPCR) of Tnfsf10 expression in mouse primary astrocytes (n = 11 vehicle; n = 3 IL-1β; n = 3 TNF; n = 3 IFNβ; n = 4 biological replicates otherwise). Unpaired two-tailed t-test. b, Levels of pSTAT1 in mouse primary astrocytes after 60-min treatment with IFNγ or vehicle (n = 4 biological replicates per condition). Experiment repeated three times. Unpaired two-tailed t-test. c, Bottom, STAT1-binding motifs (STAT1 sites 1–4; S1–S4) in the Tnfsf10 promoter. Top, STAT1 chromatin immunoprecipitation (ChIP)-qPCR after IFNγ or vehicle treatment. n = 3 per condition. Unpaired two-tailed t-test. d, qPCR of Tnfsf10 expression in mouse primary astrocytes (n = 7 vehicle, IFNγ, IFNγ + GM-CSF; n = 3 mice otherwise). Welch’s two-tailed t-test, In-normalized data. e, FACS analysis of LAMP1+TRAIL+ astrocytes (day 28 after EAE induction; n = 5 per group). Unpaired two-tailed t-test. f, EAE curves (n = 24 sgScramble; n = 18 sgIfngr1; n = 14 sgIfngr2; n = 12 sgStat1 mice). Experiment repeated three times. Two-way repeated measures ANOVA. g, TRAIL expression in astrocytes of knockdown mice (n = 9 sgScramble; n = 5 sgIfngr1; n = 5 sgIfngr2; n = 9 sgStat1 mice). Unpaired two-tailed t-test. h, FACS analysis of CNS IFNγ+IL-17+CD4+ T cells in knockdown mice (n = 6 sgScramble, n = 6 sgIfngr1, n = 5 sgIfngr2, n = 3 sgStat1 mice). Two-tailed Mann–Whitney test. i, Activation of caspase-3 in CNS CD4+T cells (n = 6 sgScramble, sgIfngr1; n = 5 mice otherwise). Unpaired two-tailed t-test. j, k, Heat map (j) and GSEA (k) of astrocyte gene expression (day 24 after EAE induction; n = 4 mice per group). l, Activation of caspase-3 in CD4+T cells co-cultured with astrocytes and anti-TRAIL blocking antibody (n = 7 biological replicates) per condition. Unpaired two-tailed t-test. m, Expression of DR5 in CD4+ T cells in peak EAE mice (day 18 after EAE induction; n = 6 mice per group). One-way ANOVA, Tukey’s post-hoc test. n, Schematic (left) and quantification (right) of active caspase-3 expression in DR5+ and DR5− CD4+ T cells isolated from peak EAE mice (day 18 after EAE induction), co-cultured with IFNγ-pre-treated astrocytes (n = 7 biological replicates per group) PI, propidium iodide. Unpaired two-tailed t-test. Data are mean ± s.e.m. (a, c, d–i, l–n).

Article Snippet: For analysis of astrocytes derived from Aldh1l1 creERT2tdTomato reporter mice, CNS cells were stained with APC anti-mouse LAMP1 (BioLegend, 121614, 1:100) and PE–Cy7 anti-mouse TRAIL (BioLegend, 109311, 1:100) for 45 min on ice.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Two Tailed Test, Binding Assay, Chromatin Immunoprecipitation, MANN-WHITNEY, Activation Assay, Cell Culture, Blocking Assay, Isolation

Journal: Nature

Article Title: Gut-licensed IFNγ + NK cells drive LAMP1 + TRAIL + anti-inflammatory astrocytes

doi: 10.1038/s41586-020-03116-4

Figure Lengend Snippet: a, STAT1 phosphorylation determined by flow cytometry in mouse astrocytes in culture treated with IFNγ or vehicle. n = 4 per condition. Unpaired two-tailed t-test. Experiment repeated three times. b, Analysis of Tnfsf10 expression in primary microglia and neurons in response to IFNγ. n = 8 for neuron IFNγ-treated, n = 5 neuron veh, n = 6 otherwise. Unpaired two-tailed t-test, data log-normalized for neuron Tnfsf10. c, Analysis of other apoptosis-promoting molecules expressed by astrocytes. n = 14 Fasl vehicle; n = 13 Gzmb vehicle; n = 12 Prf1 vehicle.; n = 7 Fasl IFNγ; n = 7 Prf1 IFNγ; n = 3 Gzmb IFNγ; n = 3 for Tnfsf10. Unpaired two-tailed t-test. Tnfsf10 data In-transformed. Note the consistent axis scale. d, Analysis of Tnfsf10 expression by qPCR in astrocytes isolated from mice null for the indicated genes 29 days after EAE induction. n = 5 Ctrl, n = 3 csf2rbf/f-Aldh1l1creERT2, n = 6 Tnfrsf1a−/−;Tnfrsflb−/−mice. Unpaired two-tailed t-test (Tnfrsf1a−/−;Tnfrsf1b−/−) and Mann–Whitney test (Csf2rbf/f-Aldh1l1creERT2). e, EAE in Ifngr1+/+ (n = 12 mice) or Ifngr1−/− (n = 10 mice). Two-way repeated measures ANOVA. f, Flow cytometry analysis of T cells in the CNS of Ifngr1+/+ or Ifngr1−/− mice. n = 5 mice per group. Unpaired two-tailed t-test. g, Caspase-3 activation in CD4+ T cells isolated from the CNS of Ifngr1+/+ or Ifngr1−/− mice. n = 6 per group. Unpaired two-tailed t-test. Data are mean ± s.e.m.

Article Snippet: For analysis of astrocytes derived from Aldh1l1 creERT2tdTomato reporter mice, CNS cells were stained with APC anti-mouse LAMP1 (BioLegend, 121614, 1:100) and PE–Cy7 anti-mouse TRAIL (BioLegend, 109311, 1:100) for 45 min on ice.

Techniques: Flow Cytometry, Two Tailed Test, Expressing, Transformation Assay, Isolation, MANN-WHITNEY, Activation Assay

Journal: Nature

Article Title: Gut-licensed IFNγ + NK cells drive LAMP1 + TRAIL + anti-inflammatory astrocytes

doi: 10.1038/s41586-020-03116-4

Figure Lengend Snippet: a, Left, immunostaining of spinal cord sections from naive and EAE mice (n = 7 sections (naive); n = 5 sections (EAE); n = 3 mice per group). Right, quantification of TRAIL expression normalized to control or parenchyma within images. Unpaired two-tailed t-test (left) and Kolmogorov–Smirnov test (right). Scale bar, 20 μm. b, Left, immunostaining of meninges from healthy control individuals (control) and from patients with MS (n = 6 sections (control); n = 5 sections (MS); n = 3 patients per group). Right, TRAIL expression quantified as in a. Unpaired two-tailed t-test (left) and Kolmogorov–Smirnov test (right). Scale bar, 20 μm. c, Immunostaining of TRAIL+ astrocytes in mouse CNS after peripheral intravenous injection of fluorescent WGA. b.v., blood vessel. Experiment repeated twice. Scale bar, 15 μm. d, RNAscope analysis of mouse spinal cord (n = 8 images (naive); n = 6 images (EAE)). Unpaired two-tailed t-test. Experiment repeated three times. Scale bar, 15 μm. e, IFNγ expression determined by flow cytometry in meninges from naive mice. NK cells, NK1.1+CD200R−; type-1 innate lymphoid cells (ILC1), NK1.1+CD200R+ (n = 4 mice (NK); n = 5 mice otherwise). One-way ANOVA, Tukey’s post-hoc test. f, Quantification of IFNγ+ NK cells in meninges from IfngEYFP mice (left) or in meninges from healthy control individuals (right) (n = 6 mice; n = 6 individuals). Unpaired two-tailed t-test. Scale bars, 100 μm. g, FACS analysis of meningeal NK cells (left) and TRAIL+ astrocytes (right) 72 h after treatment with anti-NK1.1 or isotype control antibody (n = 4 mice per group). Unpaired two-tailed t-test. h, FACS analysis of LAMP1+TRAIL+ astrocytes co-cultured with IFNγ+or IFNγ− NK cells from IfngEYFP mice (n = 3 per group). Unpaired two-tailed t-test. i, FACS analysis of IFNγ+ cells (left) and MFI (right) in meninges from naive and EAE (day 22 after EAE induction) mice. Left: n = 4 mice per group. One-way ANOVA, Tukey’s post-hoc test. Right: n = 3 naive; n = 5 EAE mice. Unpaired two-tailed t-test per cell type by condition. Data are mean ± s.e.m. (a, b, d, e–i).

Article Snippet: For analysis of astrocytes derived from Aldh1l1 creERT2tdTomato reporter mice, CNS cells were stained with APC anti-mouse LAMP1 (BioLegend, 121614, 1:100) and PE–Cy7 anti-mouse TRAIL (BioLegend, 109311, 1:100) for 45 min on ice.

Techniques: Immunostaining, Expressing, Two Tailed Test, Injection, Flow Cytometry, Cell Culture

Journal: Nature

Article Title: Gut-licensed IFNγ + NK cells drive LAMP1 + TRAIL + anti-inflammatory astrocytes

doi: 10.1038/s41586-020-03116-4

Figure Lengend Snippet: a–c, FACS analysis of LAMP1+TRAIL+ astrocytes (a), meningeal IFNγ+NK (NK1.1+CD200R−) cells (b) and meningeal NK cells (c) in SPF and germ-free (GF) mice (n = 3 SPF mice; n = 5 GF mice (a); n = 6 mice per group (b); n = 9 mice per group (c)). Unpaired two-tailed t-test. d, Schematic of adoptive transfer experiment. WT, wild type. e, f, FACS analysis of meningeal IFNγ+ NK cells (e) and LAMP1+TRAIL+ astrocytes (f) in mice receiving 1 × 106 NK cells (e: n = 4 SPF wild type; n = 5 mice otherwise; one-way ANOVA, Fisher’s test; f: n = 10 SPF wild type; n = 9 Ifng−/− mice; Kolmogorov–Smirnov two-tailed test). Ifng−/− data are log-normalized in f. g, FACS analysis of IFNγ+NK1.1+CD200R− cells in the meninges (left), total NK1.1+CD200R− cells in the meninges (middle) and TRAIL+ astrocytes in the CNS (right) of mice treated as indicated (left: n = 8 vehicle, n = 4 ABX, n = 5 ABX + FMT mice; middle: n = 10 vehicle, n = 5 mice otherwise; right: n = 10 vehicle, n = 5 mice otherwise). Two-tailed Mann–Whitney test (left, middle); unpaired two-tailed t-test (right). h, Schematic of Kaede photoconversion experiment. i, Representative analysis of splenic NK1.1+CD200R−CD3− cells after photoconversion in the small intestine. Conv, converted. j, Quantification of NK cells in meninges and CNS parenchyma after photoconversion in the small intestine (n = 4 mice not converted; n = 3 mice converted + 24 h). Unpaired two-tailed t-test. k, Quantification of splenic NK cells (left) and Ifng expression in splenic NK cells (right) in mice after small intestine photoconversion and indicated treatments (left: n = 5 mice per group; right: n = 5 vehicle; n = 9 ABX; n = 10 ABX + FMT mice). Unpaired two-tailed t-test. Data are mean ± s.e.m. (a–c, e–g, i–k).

Article Snippet: For analysis of astrocytes derived from Aldh1l1 creERT2tdTomato reporter mice, CNS cells were stained with APC anti-mouse LAMP1 (BioLegend, 121614, 1:100) and PE–Cy7 anti-mouse TRAIL (BioLegend, 109311, 1:100) for 45 min on ice.

Techniques: Two Tailed Test, Adoptive Transfer Assay, MANN-WHITNEY, Expressing

Journal: Pharmacological research

Article Title: RHBDF1 deficiency suppresses melanoma glycolysis and enhances efficacy of immunotherapy by facilitating glucose-6-phosphate isomerase degradation via TRIM32.

doi: 10.1016/j.phrs.2023.106995

Figure Lengend Snippet: Fig. 3. RHBDF1 maintains the stability of GPI in melanoma. (A) Western blotting analysis of the glycolytic enzymes′ expression after RHBDF1 knockout or knockdown in B16F10 cells and A375 cells. (B) Immunofluorescence staining of GPI protein in B16F10-EV/R1KO tumors. (Red: GPI, blue: DAPI, scale bar: 50 µm). (C) The correlation of RHBDF1 with GPI, PGAM1 and LDHA at the transcriptional level in GEPIA database. (D) qPCR analysis of GPI, PGAM1 and LDHA levels in B16F10 and A375 cells with or without RHBDF1 expression (n = 3). (E) Immunofluorescence staining of the distribution of GPI-6Myc in lysosomes in B16F10-R1KO cells. GPI-6Myc was transfected into the B16F10-R1KO cells for 24 h and the cells were treated with NH4CI for 6 h before staining, the white arrows indicate the co- location of GPI and the lysosome marker, LAMP1 (Red: Myc, green: LAMP1, blue: DAPI, scale bar: 25 µm). (F, G) Western blotting analysis of GPI protein levels in B16F10 and A375 cells when treated with NH4Cl 20 mmol/L or 3-Methyladenine (3-MA) 5 mmol/L for the indicated time. Error bars were represented as mean ± SD, two tailed Student′s t test was used for significance tests between two groups, * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Primary antibodies for Myc tag (16286–1-AP), HA tag (51064–2-AP and Chromotek, 7C9–20), PGAM1 (16126–1-AP), CD107a/LAMP1 (APC-65050) and heavy chain of rabbit IgG (P66467–1-lg) were purchased from Proteintech (Wuhan, China).

Techniques: Western Blot, Expressing, Knock-Out, Knockdown, Immunofluorescence, Staining, Transfection, Marker, Two Tailed Test

Journal: Frontiers in immunology

Article Title: ELK4 exerts opposite roles in cytokine/chemokine production and degranulation in activated mast cells.

doi: 10.3389/fimmu.2023.1171380

Figure Lengend Snippet: FIGURE 4 Elk4 deficiency promotes degranulation and histamine release. (A) Time course and dose-dependent curve detection of the beta-hexosaminidase releases. BMMCs were sensitized with anti-DNP-IgE (1 mg/ml) overnight, then stimulated with DNP-HSA (10, 100, 1000 ng/ml) for 60min (Left) or stimulated with DNP-HSA (100 ng/ml) for 5min, 15min, 30min and 60min (Right). Bar, mean; error bar, SD; n=4; **p<0.01. (B) The total contents of beta-hexosaminidase in Elk4 WT, HZ and KO BMMCs are shown. Bar, mean; error bar, SD; n=3; *p < 0.05; **p<0.01. (C, D) Degranulation of Elk4 WT, HZ and KO PCMCs was assessed by beta-hexosaminidase release assay. PCMCs were sensitized with anti-DNP-IgE (1 mg/ml) overnight and stimulated with DNP-HSA (100 ng/ml) for 1 hour. The released contents (A) and the total contents (B) of beta-hexosaminidase in Elk4 WT, HZ and KO PCMCs are shown. Bar, mean; error bar, SD; n=2; **p<0.01. (E, F) Degranulation of Elk4 WT, HZ and KO BMMCs (E) and PCMCs (F) was assessed by cell-surface LAMP1 staining using flow cytometry. Both BMMCs and PCMCs were sensitized with anti-DNP-IgE (1 mg/ml) overnight and stimulated with DNP-HSA (100 ng/ml) for 1 hour before LAMP1 staining. Bar, mean; error bar, SD; n=3; *p<0.05.

Article Snippet: Antibodies for flow cytometry were as follows: anti-mouse CD45-violetFluor450 (Multi Sciences, AM04512), anti-mouse CD45-PECY7 (BioLegend, 103114), anti-mouse CD19-FITC (Multi Sciences, AM01901), anti-mouse CD3-PE (BioLegend, 100205), anti-mouse CD11b-Percp-cy5.5 (Multi Sciences, AH011B07), anti-mouse GR-1-PE (Multi Sciences, AM0L604), Lamp1-FITC (ProteinTech, FITC-65050), IL-6-eFluor450 (ebioscience, 48-7061-82), CCL3-PE(ebioscience, 12-7532-82), TNFa-APC(ebioscience, 17-7321-82). b-Hexosaminidase assay BMMCs and PCMCs were sensitized with anti-DNP-IgE (1 mg/ml) overnight.

Techniques: Release Assay, Staining, Cytometry