Journal: Frontiers in Immunology

Article Title: Cochlear Marginal Cell Pyroptosis Is Induced by Cisplatin via NLRP3 Inflammasome Activation

doi: 10.3389/fimmu.2022.823439

Figure Lengend Snippet: Cisplatin induced an increased expression of NLRP3 in MCs at both the mRNA level and the protein level. (A) The expression difference of NLRP3 mRNA in MCs between the control and the cisplatin group (5 μmol/l cisplatin for 24 h). (B, C) The increased expression of NLRP3 protein in the cisplatin group compared with the control detected by immunofluorescence (B) and Western blotting (C) . Green fluorescence indicates FITC-labeled NLRP3 protein, and blue fluorescence indicates DAPI-labeled nuclei. Scale bars: 50 µm. Representative results of at least three repeated experiments are shown. *p < 0.05, ***p < 0.001.

Article Snippet: For the detection of target proteins, the cells were washed after permeabilization, blocked with donkey serum (AntGene, Wuhan, China) for 30 min at room temperature, then incubated with a primary antibody against CK-18 (abs123946, 1:400, Absin Bioscience, Shanghai, China), NLRP3 (NBP2-12446, 1:100, Novus Biologicals, Littleton, CO, USA), ASC (ab175449, 1:100, Abcam, Cambridge, MA, USA), caspase-1 (sc-398715, 1:100, Santa Cruz, Dallas, TX, USA), thioredoxin-interacting protein (TXNIP, ab210826, 1:300, Abcam), and GSDMD (20770-1-AP, 1:500, Proteintech) overnight at 4°C.

Techniques: Expressing, Immunofluorescence, Western Blot, Fluorescence, Labeling

Journal: Frontiers in Immunology

Article Title: Cochlear Marginal Cell Pyroptosis Is Induced by Cisplatin via NLRP3 Inflammasome Activation

doi: 10.3389/fimmu.2022.823439

Figure Lengend Snippet: Downregulation of NLRP3 decreased the number of MCs with cell membrane rupture (A) or DNA rupture (B) . (A) The differences in the percentage of MCs with cell membrane rupture (the cells in the Q1 and Q2 quadrants) between the groups. (B) The differences in the percentage of MCs with DNA fracture (TUNEL positive) between the groups. Blue fluorescence indicates DAPI-labeled cell nuclei, and red fluorescence indicates DNA fragmentation labeled with Alexa Fluor 640. The red fluorescence overlapping with the nucleus was judged to be TUNEL-positive cells (white arrows). Scale bar: 100 µm. Representative results of at least three repeated experiments are shown. **p < 0.01, ***p < 0.001.

Article Snippet: For the detection of target proteins, the cells were washed after permeabilization, blocked with donkey serum (AntGene, Wuhan, China) for 30 min at room temperature, then incubated with a primary antibody against CK-18 (abs123946, 1:400, Absin Bioscience, Shanghai, China), NLRP3 (NBP2-12446, 1:100, Novus Biologicals, Littleton, CO, USA), ASC (ab175449, 1:100, Abcam, Cambridge, MA, USA), caspase-1 (sc-398715, 1:100, Santa Cruz, Dallas, TX, USA), thioredoxin-interacting protein (TXNIP, ab210826, 1:300, Abcam), and GSDMD (20770-1-AP, 1:500, Proteintech) overnight at 4°C.

Techniques: TUNEL Assay, Fluorescence, Labeling

Journal: Frontiers in Immunology

Article Title: Cochlear Marginal Cell Pyroptosis Is Induced by Cisplatin via NLRP3 Inflammasome Activation

doi: 10.3389/fimmu.2022.823439

Figure Lengend Snippet: Downregulation of NLRP3 reversed the cisplatin-induced ultramicroscopic changes in MCs. (A) The morphologic changes of MCs after different treatments under TEM. The red arrows indicate the ruptured cell membrane and local lighter-stained cytoplasm. Scale bars: 2 µm. (B) The morphological changes of MCs observed by SEM. The red arrows indicate the withered tree-like cell membrane at the edge of the cell. Scale bars: 50 µm. Representative results of at least three repeated experiments are shown.

Article Snippet: For the detection of target proteins, the cells were washed after permeabilization, blocked with donkey serum (AntGene, Wuhan, China) for 30 min at room temperature, then incubated with a primary antibody against CK-18 (abs123946, 1:400, Absin Bioscience, Shanghai, China), NLRP3 (NBP2-12446, 1:100, Novus Biologicals, Littleton, CO, USA), ASC (ab175449, 1:100, Abcam, Cambridge, MA, USA), caspase-1 (sc-398715, 1:100, Santa Cruz, Dallas, TX, USA), thioredoxin-interacting protein (TXNIP, ab210826, 1:300, Abcam), and GSDMD (20770-1-AP, 1:500, Proteintech) overnight at 4°C.

Techniques: Staining

Journal: Frontiers in Immunology

Article Title: Cochlear Marginal Cell Pyroptosis Is Induced by Cisplatin via NLRP3 Inflammasome Activation

doi: 10.3389/fimmu.2022.823439

Figure Lengend Snippet: The expression differences of key molecules in NLRP3 inflammasome-mediated pyrolysis (NLRP3, ASC, caspase-1, IL-1β, and GSDMD) in mRNA level (A) and in protein level (B) in the control, cisplatin, cisplatin+siNC, and cisplatin+siNLRP3 groups. (C) The difference in the IL-1β concentrations in the supernatant of each group. Representative results of at least three repeated experiments are shown. *p < 0.05, **p < 0.01, ***p < 0.001. ns, no significance.

Article Snippet: For the detection of target proteins, the cells were washed after permeabilization, blocked with donkey serum (AntGene, Wuhan, China) for 30 min at room temperature, then incubated with a primary antibody against CK-18 (abs123946, 1:400, Absin Bioscience, Shanghai, China), NLRP3 (NBP2-12446, 1:100, Novus Biologicals, Littleton, CO, USA), ASC (ab175449, 1:100, Abcam, Cambridge, MA, USA), caspase-1 (sc-398715, 1:100, Santa Cruz, Dallas, TX, USA), thioredoxin-interacting protein (TXNIP, ab210826, 1:300, Abcam), and GSDMD (20770-1-AP, 1:500, Proteintech) overnight at 4°C.

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Cochlear Marginal Cell Pyroptosis Is Induced by Cisplatin via NLRP3 Inflammasome Activation

doi: 10.3389/fimmu.2022.823439

Figure Lengend Snippet: The expression differences of NLRP3, ASC, caspase-1, and GSDMD in the control, cisplatin, cisplatin+siNC, and cisplatin+siNLRP3 groups. Blue fluorescence indicates DAPI-labeled cell nuclei, and green fluorescence indicates FITC-labeled target proteins. Representative results of at least three repeated experiments are shown. Scale bar: 100 µm.

Article Snippet: For the detection of target proteins, the cells were washed after permeabilization, blocked with donkey serum (AntGene, Wuhan, China) for 30 min at room temperature, then incubated with a primary antibody against CK-18 (abs123946, 1:400, Absin Bioscience, Shanghai, China), NLRP3 (NBP2-12446, 1:100, Novus Biologicals, Littleton, CO, USA), ASC (ab175449, 1:100, Abcam, Cambridge, MA, USA), caspase-1 (sc-398715, 1:100, Santa Cruz, Dallas, TX, USA), thioredoxin-interacting protein (TXNIP, ab210826, 1:300, Abcam), and GSDMD (20770-1-AP, 1:500, Proteintech) overnight at 4°C.

Techniques: Expressing, Fluorescence, Labeling

Journal: Frontiers in Immunology

Article Title: Cochlear Marginal Cell Pyroptosis Is Induced by Cisplatin via NLRP3 Inflammasome Activation

doi: 10.3389/fimmu.2022.823439

Figure Lengend Snippet: The effect of downregulation of TXNIP on cisplatin-induced NLRP3 inflammasome-mediated pyrolysis-related protein expression in MCs at mRNA level (A) and protein level (B) . (C) The difference in the concentrations of IL-1β in the supernatant in each group. (D) The variation of NLRP3 expression before and after TXNIP-siRNA transfection. Scale bars: 100 µm. Representative results of at least three repeated experiments are shown. *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: For the detection of target proteins, the cells were washed after permeabilization, blocked with donkey serum (AntGene, Wuhan, China) for 30 min at room temperature, then incubated with a primary antibody against CK-18 (abs123946, 1:400, Absin Bioscience, Shanghai, China), NLRP3 (NBP2-12446, 1:100, Novus Biologicals, Littleton, CO, USA), ASC (ab175449, 1:100, Abcam, Cambridge, MA, USA), caspase-1 (sc-398715, 1:100, Santa Cruz, Dallas, TX, USA), thioredoxin-interacting protein (TXNIP, ab210826, 1:300, Abcam), and GSDMD (20770-1-AP, 1:500, Proteintech) overnight at 4°C.

Techniques: Expressing, Transfection

Journal: Frontiers in Immunology

Article Title: Cochlear Marginal Cell Pyroptosis Is Induced by Cisplatin via NLRP3 Inflammasome Activation

doi: 10.3389/fimmu.2022.823439

Figure Lengend Snippet: Downregulation of TXNIP reversed the cisplatin-induced ultramicroscopic changes in MCs, similar to that after downregulating NLRP3. (A) The morphologic changes of MCs after different treatments were evaluated under TEM. The red arrows indicate the ruptured cell membrane and local lighter-stained cytoplasm. Scale bars: 2 µm. (B) The morphological changes of MCs were observed by SEM. The red arrows indicate the withered tree-like cell membrane at the edge of the cell. Scale bars: 15 µm. Representative results of at least three repeated experiments are shown.

Article Snippet: For the detection of target proteins, the cells were washed after permeabilization, blocked with donkey serum (AntGene, Wuhan, China) for 30 min at room temperature, then incubated with a primary antibody against CK-18 (abs123946, 1:400, Absin Bioscience, Shanghai, China), NLRP3 (NBP2-12446, 1:100, Novus Biologicals, Littleton, CO, USA), ASC (ab175449, 1:100, Abcam, Cambridge, MA, USA), caspase-1 (sc-398715, 1:100, Santa Cruz, Dallas, TX, USA), thioredoxin-interacting protein (TXNIP, ab210826, 1:300, Abcam), and GSDMD (20770-1-AP, 1:500, Proteintech) overnight at 4°C.

Techniques: Staining

Journal: Frontiers in Immunology

Article Title: Cochlear Marginal Cell Pyroptosis Is Induced by Cisplatin via NLRP3 Inflammasome Activation

doi: 10.3389/fimmu.2022.823439

Figure Lengend Snippet: The schematic diagram of cisplatin-induced NLRP3 inflammasome-mediated MC pyroptosis.

Article Snippet: For the detection of target proteins, the cells were washed after permeabilization, blocked with donkey serum (AntGene, Wuhan, China) for 30 min at room temperature, then incubated with a primary antibody against CK-18 (abs123946, 1:400, Absin Bioscience, Shanghai, China), NLRP3 (NBP2-12446, 1:100, Novus Biologicals, Littleton, CO, USA), ASC (ab175449, 1:100, Abcam, Cambridge, MA, USA), caspase-1 (sc-398715, 1:100, Santa Cruz, Dallas, TX, USA), thioredoxin-interacting protein (TXNIP, ab210826, 1:300, Abcam), and GSDMD (20770-1-AP, 1:500, Proteintech) overnight at 4°C.

Techniques:

Journal: The Journal of Clinical Investigation

Article Title: P2X7R mutation disrupts the NLRP3-mediated Th program and predicts poor cardiac allograft outcomes

doi: 10.1172/JCI94524

Figure Lengend Snippet: (A) P2X7R and NLRP3 immunoprecipitation (IP) in human CD4+ T cells. Expression of NLRP3 (top blot) and P2X7R (bottom blot) is shown. Lane 1: Total protein. Lane 2: IP with NLRP3 Ab. Lane 3: IP with P2X7R Ab. Lane 4: IP with Ab alone (NLRP3 and P2X7R). Lane 5: IP with control IgG (for NLRP3 Ab in top blot, for P2X7R Ab in bottom blot). The experiment was run in triplicate (representative blot shown). (B and C) Confocal microscopy analysis (B, scale bar: 5 μm, ×100 original magnification; C, scale bars: 20 μm, ×40 original magnification) depicting baseline colocalization of P2X7R (green) and NLRP3 (red) in human CD4+ T cells. Cells were stained with DAPI (blue) and immunolabeled with anti-P2X7R (green) and anti-NLRP3 Abs (red) (n = 3). (D–F) Bar graphs depicting expression of NLRP3 mRNA by qRT-PCR (D), and protein by flow cytometry (E) and ELISA (F), evaluated in human CD4+ T cells activated with benzoyl ATP (BzATP) and treated with CE-224,535, a P2X7R inhibitor. Experiments were run in duplicate (n = 5). (G) Bar graph representing expression of NLRP3 on human CD4+P2X7R+ cells analyzed by flow cytometry upon BzATP stimulation (n = 5). (H) Representative flow dot plots of NLRP3 expression upon gating on human BzATP-stimulated CD4+P2X7R+ cells. (I) Confocal analysis (scale bar: 5 μm; ×100 original magnification) depicting colocalization of P2X7R (green) and NLRP3 (red) in CD4+ T cells upon in vitro stimulation of P2X7R with BzATP (n = 3). (J–M) Bar graphs comparing expression of NLRP3 downstream signaling Th2-related factors IL-4 (J), IRF4 (K), GATA-3 (L), and IL-10 (M) by qRT-PCR using mRNA isolated from human CD4+ T cells activated with BzATP and treated with the P2X7R inhibitor CE-224,535. Experiments were run in triplicate (n = 5). Data are expressed as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; 1-way ANOVA with Bonferroni’s post hoc test or Student’s t test. mRNA expression was normalized to β-actin (ACTB).

Article Snippet: Purified anti–human NLRP3 (IC7578G) and Alexa Fluor 488–conjugated anti–rabbit NLRP3 (IC7578G) were purchased from R&D Systems and BD Biosciences, respectively.

Techniques: Immunoprecipitation, Expressing, Control, Confocal Microscopy, Staining, Immunolabeling, Quantitative RT-PCR, Flow Cytometry, Enzyme-linked Immunosorbent Assay, In Vitro, Isolation

Journal: The Journal of Clinical Investigation

Article Title: P2X7R mutation disrupts the NLRP3-mediated Th program and predicts poor cardiac allograft outcomes

doi: 10.1172/JCI94524

Figure Lengend Snippet: (A) A 3D representation of the full-length structure of P2X7R, highlighting the putative location of the P2X7R mutation in the C-terminal intracellular portion. (B and C) Quantification of P2X7R total protein (B, ELISA, n = 3) and of P2X7R mRNA (C, qRT-PCR, n = 10) on CD4+ T cells of carrier and noncarrier patients. Samples were run in duplicate (B) or in triplicate (C) and normalized to expression level of β-actin (ACTB). (D) Transcriptome profiling of immune-relevant genes (see also Supplemental Table 3) examined in CD4+ T cells of carrier and noncarrier cardiac-transplanted patients (n = 5). (E–G) Expression of NLRP3 mRNA using qRT-PCR (E) and NLRP3 protein using flow cytometry (F) and ELISA (G) in CD4+ T cells of carrier and noncarrier patients (n = 5). (H and I) Flow cytometric expression of NLRP3 on CD4+P2X7R+ cells of carrier patients stimulated with BzATP (n = 5). (J) Percentage of P2X7R+NLRP3+ cells of carrier and noncarrier patients analyzed by immunofluorescence (Figure 1C and Supplemental Figure 2G) (n = 3). (K) Confocal microscopy analysis (×100 original magnification) of P2X7R (green) and NLRP3 (red) coexpression in CD4+ T cells of carrier patients (n = 3). Scale bar: 5 μm. (L) Subcellular localization of NLRP3 in CD4+ T cells of carrier and of noncarrier patients (n = 3). (M and N) IL-4 (M) and IRF4 (N) gene expression detected after ChIP with NLRP3 antibody in CD4+ T cells. (n = 3). (O) Quantification of NLRP3 protein measured in CD4+ T cells treated with the ubiquitin/protease inhibitor MG132 (n = 3). Bars represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; Student’s t test or 2-way ANOVA with Bonferroni’s post hoc test.

Article Snippet: Purified anti–human NLRP3 (IC7578G) and Alexa Fluor 488–conjugated anti–rabbit NLRP3 (IC7578G) were purchased from R&D Systems and BD Biosciences, respectively.

Techniques: Mutagenesis, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Expressing, Flow Cytometry, Immunofluorescence, Confocal Microscopy, Gene Expression, Ubiquitin Proteomics, Protease Inhibitor

Journal: The Journal of Clinical Investigation

Article Title: P2X7R mutation disrupts the NLRP3-mediated Th program and predicts poor cardiac allograft outcomes

doi: 10.1172/JCI94524

Figure Lengend Snippet: (A) Percentage of in vitro–generated Th17 cells obtained from CD4+ T cells of carrier and noncarrier patients (n = 8). (B and C) Representative flow zebra plots (B) and quantitative histogram (C) depicting the percentage of peripheral CD4+IL-17+ cells (n = 8). (D) IL-17 plasma levels of carrier and noncarrier patients (n = 10). (E) IL-17 levels (Luminex) measured in the supernatants of unstimulated 24-hour-cultured CD4+ T cells of carrier and noncarrier patients (n = 5). (F) Table summarizing the secretome profile (Luminex, n = 5) and primary phenotypic characteristics (flow cytometry, n = 4) of carrier and noncarrier polarized Th17 cells. (G and H) Normalized mRNA expression of Th2-related factors IL-4 (G) and GATA-3 (H) measured in noncarrier CD4+ T cells exposed to transient knockdown of NLRP3 using silencing RNA (siRNA), before and after anti-CD3-Ig/anti-CD28-Ig stimulation (n = 3). (I–K) Normalized mRNA expression of the Th2-related factors IL-4 (I), IL-10 (J), and GATA-3 (K) measured in noncarrier CD4+ T cells exposed to transient knockdown of NLRP3 (siRNA), upon BzATP exposure (n = 4). (L and M) Normalized mRNA expression of the Th2-related factors IL-4 (L) and GATA-3 (M) measured in carrier CD4+ T cells, in which NLRP3 was overexpressed, before and after anti-CD3-Ig/anti-CD28-Ig stimulation (n = 3). (N) Effects of various treatments (anti–IL-17 antibody, RMT1-10, cyclosporin A [CsA] and rapamycin [Rapa]) on in vitro–generated Th17 cells (n = 5). Experiments were run in triplicate (D, G, H, and N) or in duplicate (F and I–M). mRNA expression was normalized to ACTB. Bars represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; Student’s t test or 1-way ANOVA with Bonferroni’s post hoc test.

Article Snippet: Purified anti–human NLRP3 (IC7578G) and Alexa Fluor 488–conjugated anti–rabbit NLRP3 (IC7578G) were purchased from R&D Systems and BD Biosciences, respectively.

Techniques: In Vitro, Generated, Clinical Proteomics, Luminex, Cell Culture, Flow Cytometry, Expressing, Knockdown

Journal: The Journal of Clinical Investigation

Article Title: P2X7R mutation disrupts the NLRP3-mediated Th program and predicts poor cardiac allograft outcomes

doi: 10.1172/JCI94524

Figure Lengend Snippet: (A) P2X7R–/– mice receiving bm12 heart transplantation demonstrated reduced graft survival as compared with B6 recipients (**P < 0.01), which was significantly prolonged by anti–IL-17 treatment (murine IL-17–depleting antibody) (*P < 0.05 vs. P2X7R–/–) (n = 10 mice per group). (B–D) Semiquantification of graft infiltration (B), coronary vasculopathy (C), and myocyte necrosis (D) confirmed accelerated allograft rejection in P2X7R–/– mice (n = 3). (E) Representative H&E staining (x20 original magnification) showing graft cell infiltration (top panels), vasculopathy (middle panels), and myocyte necrosis (bottom panels) in B6 and P2X7R–/– mice. Scale bars: 200 μm (middle panels), 300 μm (top and bottom panels). (F and G) Numbers of IFN-γ–producing (F) and IL-4–producing (G) cells (ELISPOT) measured in cardiac-transplanted mice (n = 3). (H–M) Percentage of CD4+IL-17+ (H), CD4+IFN-γ+ (I), CD4+IL-10+ (J), CD4+CD44hiCD62Llo (K), CD8+CD44hiCD62Llo (L), and CD4+CD25+Foxp3+ (M) cells detected by flow cytometry in B6 and P2X7R–/– cardiac-transplanted mice and in P2X7R–/– anti–IL-17–treated mice (n = 5). (N) Serum IL-17 level (Luminex) measured in B6 and P2X7R–/– cardiac-transplanted mice and in P2X7R–/– anti–IL-17–treated mice (n = 5). (O) Percentage of CD4+NLRP3+ cells analyzed by flow cytometry in P2X7R–/– and B6 mice (n = 3). (P) Number of IL-4–producing cells (ELISPOT) in P2X7R–/– and B6 mice upon allostimulation (n = 3). (Q) Serum IL-4 level (Luminex), measured in B6 and P2X7R–/– cardiac-transplanted mice (n = 5). Samples were run in duplicate (Luminex) and in triplicate (ELISPOT). Bars represent mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.001; log-rank (Mantel-Cox) test (A), Wilcoxon’s and Student’s t test (2 groups), 1-way ANOVA with Bonferroni’s post hoc test (3 groups).

Article Snippet: Purified anti–human NLRP3 (IC7578G) and Alexa Fluor 488–conjugated anti–rabbit NLRP3 (IC7578G) were purchased from R&D Systems and BD Biosciences, respectively.

Techniques: Transplantation Assay, Staining, Enzyme-linked Immunospot, Flow Cytometry, Luminex

Journal: The Journal of Clinical Investigation

Article Title: P2X7R mutation disrupts the NLRP3-mediated Th program and predicts poor cardiac allograft outcomes

doi: 10.1172/JCI94524

Figure Lengend Snippet: (A) Bar graph depicting the percentage of cardiac-transplanted patients who carry the WT or mutant P2X7R allele (n = 102) with an MIT change greater than 0.5 mm (early cardiac allograft vasculopathy, in black) at 1 year after transplantation in the CTOT-05 cohort. (B) Bar graph depicting the number of acute rejection episodes in cardiac-transplanted patients who carry the WT (black) or mutant (white) P2X7R allele (n = 181) within the first year after transplant in the NIT-Bergamo cohort. (C) Bar graph depicting the percentage of cardiac-transplanted patients who carry the WT or mutant P2X7R allele (n = 130) with major adverse cardiac events (MACEs, in black) at 10 years of follow-up in the AIRT-Bologna cohort. In A and C: black, percentage of patients who experienced the event; white, percentage who were free from events. (D) Line graph depicting the estimated odds ratio (OR) for clinical outcomes recorded in the 3 cohorts of cardiac-transplanted patients who carry the WT or mutant P2X7R allele. In the NIT-Bergamo cohort, the OR was calculated based on the requirement of medical intervention for acute rejection episodes with a frequency of greater or less than 3 episodes. *P < 0.05; **P < 0.01. Supplemental Tables 7–9 report detailed analyses. Fisher’s exact and Student’s t tests. (E and F) A stable connection between P2X7R and NLRP3 is necessary to establish a physiological NLRP3-mediated Th2 program (E), while alteration in the P2X7R intracellular domain induces NLRP3 displacement and retains NLRP3 in the cell membrane, thus preventing its nuclear activity and accelerating ubiquitination of NLRP3 (F). This shifts the balance of the immune response toward Th17 cells and favors the development of immune-related events, such as allograft rejection and vasculopathy. Ub, ubiquitin; eATP, extracellular ATP.

Article Snippet: Purified anti–human NLRP3 (IC7578G) and Alexa Fluor 488–conjugated anti–rabbit NLRP3 (IC7578G) were purchased from R&D Systems and BD Biosciences, respectively.

Techniques: Mutagenesis, Transplantation Assay, Membrane, Activity Assay, Ubiquitin Proteomics

Journal: Nature Communications

Article Title: NLRP3 inflammasome in fibroblasts links tissue damage with inflammation in breast cancer progression and metastasis

doi: 10.1038/s41467-019-12370-8

Figure Lengend Snippet: The NLRP3 inflammasome pathway is upregulated in fibroblasts during mammary carcinogenesis. a Scheme of fibroblast isolation procedure. b Principal component analysis (PCA) of samples included in the microarray transcriptome analysis. Each focal point represents a cohort of mice. Normal n = 6 mice/cohort, Hyperplasia n = 6 mice/cohort, Carcinoma n = 4 mice/cohort, advanced carcinoma (A. Carcinoma) n = 3 mice/cohort. c Venn diagrams depicting overlap of differentially expressed genes in fibroblasts isolated from distinct stages of MMTV-PyMT mammary carcinoma. d Pro-inflammatory genes upregulated in CAFs isolated from MMTV-PyMT tumours. Inflammasome-related genes are highlighted. e Heat map of Nlrp3/Il1b pathway-related genes. f qRT-PCR analysis of Nlrp3/Il1b pathway related genes in fibroblasts isolated by FACS (PDGFRα + CD45 − EpCAM − ) from MMTV-PyMT mice or normal mammary glands. Data are presented as mean ± s.d of technical repeats; One-way analysis of variance followed by Tukey’s test. * p < 0.05, ** p < 0.005, *** p < 0.0005, n = pools of 3 mice/group. Results are representative of three independent biological experiments. g Representative IHC staining of NLRP3 in PyMT tumours or in normal mammary glands. Arrows indicate fibroblasts. Scale bar, 25 µm. h Quantification of staining shown in g . Multiple fields of 3 mice/group were analysed for the percentage of NLRP3 expressing fibroblasts out of total fibroblasts. Data are presented as mean ± s.e.m; Mann–Whitney test. Source data are provided as a Source Data file

Article Snippet: The intracellular staining for NLRP3 was performed with APC-conjugated antibody (R&D, IC7578A, diluted 1:10) following fixation and permeabilization of cells using BD Cytofix/Cytoperm kit (BD Bioscience, 554714) according to the manufacturer’s protocol.

Techniques: Isolation, Microarray, Quantitative RT-PCR, Immunohistochemistry, Staining, Expressing, MANN-WHITNEY

Journal: Nature Communications

Article Title: NLRP3 inflammasome in fibroblasts links tissue damage with inflammation in breast cancer progression and metastasis

doi: 10.1038/s41467-019-12370-8

Figure Lengend Snippet: The NLRP3 inflammasome pathway is up-regulated in CAFs in human breast tumours. a – d Expression levels of Nlrp3/Il1b pathway-related genes in tumour-associated stroma of breast cancer patients with infiltrating ductal carcinoma (IDC) staged 1/2 vs. 3 ( n = 53) or in normal breast stroma ( n = 6). Data was obtained from NCBI GEO (Dataset accession number GSE 9014) and is presented as mean ± s.e.m.; One-way analysis of variance test followed by Tukey’s multiple comparisons test. e – g Analysis of NLRP3 expression in fibroblasts in human IDC or in normal breast tissue sections. Images were obtained from the Human Protein Atlas. e Representative images, arrows in insets indicate fibroblasts. f Quantification of the percentage of NLRP3-expressing fibroblasts out of total fibroblasts for each sample. n = 2 samples for normal and 8 samples for IDC, error bars represent s.e.m; Welch’s t -test. g Quantification of staining intensity for NLRP3 in fibroblasts shown in e . h – j Staining for IL-1β in benign or malignant (DCIS/IDC) tissue sections from human breast cancer patients. h Representative images of stained benign and IDC breast tissue sections. Arrows indicate fibroblasts. Scale bar, 50 µm. i , j Quantification of the abundance i and staining intensity j of labelled fibroblasts, n = 73 (benign: n = 31, DCIS: n = 16, IDC: n = 26); Mann–Whitney test ( i ) or Kruskal–Wallis test followed by Dunn’s multiple comparisons test ( j ). Data are presented as mean ± s.e.m; Mann–Whitney test. Source data are provided as a Source Data file

Article Snippet: The intracellular staining for NLRP3 was performed with APC-conjugated antibody (R&D, IC7578A, diluted 1:10) following fixation and permeabilization of cells using BD Cytofix/Cytoperm kit (BD Bioscience, 554714) according to the manufacturer’s protocol.

Techniques: Expressing, Staining, MANN-WHITNEY

Journal: Nature Communications

Article Title: NLRP3 inflammasome in fibroblasts links tissue damage with inflammation in breast cancer progression and metastasis

doi: 10.1038/s41467-019-12370-8

Figure Lengend Snippet: Fibroblasts function as DAMP sensors via NLRP3 inflammasome signalling. a – e Primary FVB/n NMFs were incubated for 24 h in control medium or in medium containing one of the following DAMPs: ATP 1 mM, H 2 O 2 300 μM, MSU 5 μg/mL, 5% necrotic fluid (extracted from late-stage PyMT tumours). Nlrp3 a and Il1b b expression in fibroblasts were analysed by qRT-PCR. Data are presented as mean ± s.d.; One-way analysis of variance test followed by Dunnett’s multiple comparisons test. Representative of three independent experiments. c Caspase-1 processing was assessed by western blot of cell lysates with anti-Caspase-1. β-actin was utilised as a loading control. The samples shown are derived from the same experiment. Data are representative of three independent experiments. d ELISA quantification of IL-1β secretion. Data are presented as mean ± s.d. of biological replicates ( n = 4 per group); Kruskal–Wallis test followed by Dunn’s multiple comparisons test. Representative of three independent experiments. e Primary FVB/n NMFs were incubated for 24 h with 5% necrotic fluid or control medium and expression of selected genes was analysed using qRT-PCR. Data are presented as mean ± s.d. of biological replicates ( n = 3 per group); Welch’s t -test. Representative of two independent experiments. f Schematic illustration of cutaneous wound assay. Cutaneous wounds were generated in 8 weeks old FVB/n mice using the dermal punch method. Wounded or control skin fibroblasts were isolated by FACS as PDGFRα + CD45 − EpCAM − cells. g qRT-PCR analysis of the expression of selected genes in wound-derived fibroblasts as compared to their expression in fibroblasts from normal skin. n = pool of 8 mice per group. Data are presented as mean ± s.d. of biological repeats; Welch’s t -test. Representative of three independent biological experiments. Source data are provided as a Source Data file

Article Snippet: The intracellular staining for NLRP3 was performed with APC-conjugated antibody (R&D, IC7578A, diluted 1:10) following fixation and permeabilization of cells using BD Cytofix/Cytoperm kit (BD Bioscience, 554714) according to the manufacturer’s protocol.

Techniques: Incubation, Expressing, Quantitative RT-PCR, Western Blot, Derivative Assay, Enzyme-linked Immunosorbent Assay, Generated, Isolation

Journal: Nature Communications

Article Title: NLRP3 inflammasome in fibroblasts links tissue damage with inflammation in breast cancer progression and metastasis

doi: 10.1038/s41467-019-12370-8

Figure Lengend Snippet: CAF-derived NLRP3 inflammasome signalling facilitates tumour growth. a Scheme of experiments analysed in b – h . shNlrp3 AT3 cells were orthotopically co-injected with WT ( Nlrp3 +/+ ) or with Nlrp3 −/− NMFs (C57BL/6) into C57BL/6 mice. b Growth curves of AT3 tumours. n = 7 mice per group. c Tumour weights at termination of experiment. n = 6 and 7 mice per group (WT and Nlrp3 −/− , respectively). d Flow cytometry analysis of Gr1 + cell infiltration into AT3 tumours co-injected with WT NMFs ( n = 4) or with Nlrp3 −/− NMFs ( n = 3). Data presented are percentage of CD45 + cells, normalised to WT. e Representative images of staining with anti-Gr1 antibody in AT3 tumours. Scale bar, 100 μm. f Quantification of staining shown in d . Three fields of 3 mice/group were analysed; Data are presented as mean Gr1 + cells/field ± s.d; Mann–Whitney test. g , h Flow cytometry analysis of CD11b + Ly6C high Ly6G − and CD11b + Ly6G + Ly6C − cell infiltration into AT3 tumours co-injected with WT NMFs ( n = 4) or with Nlrp3 −/− NMFs ( n = 3). Data presented are percentage of CD45 + cells, normalised to WT. i Scheme of experiments analysed in j – r . Met-1 cells were orthotopically co-injected with NMFs depleted of Nlrp3 or Il1b expression (shNlrp3, and shIl1b), or with control NMFs (shScramble). j Growth curve and k weight at endpoint of Met-1 tumours. n = 5, 10, 20, 19 tumours (TCs only, shScramble, shNlrp3, and shIl1b, respectively). l – n Flow cytometry analysis of Gr1 + cell infiltration into Met-1 tumours. n = 7, 14, and 16 per group (shScramble, shNlrp3, and shIl1b, respectively). Data presented are percentage of CD45 + CD11b + cells, normalised to shScramble. o – r Met-1 tumours were analysed for the expression of selected genes using qRT-PCR. n = 6, 7, and 5–6 tumours per group (shScramble, shNlrp3, and shIl1b, respectively). Data are presented as fold change from shScramble. In b – d , g , h , j , k , data are presented as mean ± s.e.m; Welch’s t -test, * p < 0.05, ** p < 0.005. In j – r data are presented as mean ± s.e.m; One-way analysis of variance test followed by Tukey’s multiple comparisons test. Data are representative of three independent biological experiments. Source data are provided as a Source Data file

Article Snippet: The intracellular staining for NLRP3 was performed with APC-conjugated antibody (R&D, IC7578A, diluted 1:10) following fixation and permeabilization of cells using BD Cytofix/Cytoperm kit (BD Bioscience, 554714) according to the manufacturer’s protocol.

Techniques: Derivative Assay, Injection, Flow Cytometry, Staining, MANN-WHITNEY, Expressing, Quantitative RT-PCR

Journal: Nature Communications

Article Title: NLRP3 inflammasome in fibroblasts links tissue damage with inflammation in breast cancer progression and metastasis

doi: 10.1038/s41467-019-12370-8

Figure Lengend Snippet: Summary. Activation of the NLRP3 inflammasome in cancer-associated fibroblasts links tissue damage with tumour-promoting inflammation in breast cancer progression and metastasis. Figure preparation partially involved elements from the Servier Medical Art. Figure preparation involved using elements from the Servier Medical Art and somersault18:24 (somersault1824.com)

Article Snippet: The intracellular staining for NLRP3 was performed with APC-conjugated antibody (R&D, IC7578A, diluted 1:10) following fixation and permeabilization of cells using BD Cytofix/Cytoperm kit (BD Bioscience, 554714) according to the manufacturer’s protocol.

Techniques: Activation Assay