p2rx1 knockout (ko) mice (p2rx1−/)  (GemPharmatech Co Ltd)

Journal: Cell Biology and Toxicology

Article Title: P2rx1 deficiency alleviates acetaminophen-induced acute liver failure by regulating the STING signaling pathway

doi: 10.1007/s10565-023-09800-1

Figure Lengend Snippet: Knockout of P2rx1 alleviates liver injury in the AILI mouse model. Dynamic hepatic relative P2rx1 mRNA expression and P2RX1 expression in (a) DILI patients and (b) APAP-treated WT mice (n = 4–6 per group); (c) Representative IF staining images of P2RX1 in WT mice with or without AILI (origin magnification × 100, scale bar = 100 μm); (d) Serum ALT and AST levels in WT and P2rx1 −/− mice after a single dose of PBS or APAP (300 mg/kg, n = 4–6 per group); (e) Representative images of H&E staining in liver sections of WT and P2rx1 −/− mice with or without APAP challenge (origin magnification × 100). Quantification of necrotic areas in liver sections by H&E staining (n = 4–6 per group); (f) Survival curve of WT and P2rx1 −/− mice in response to a single lethal dose of APAP (500 mg/kg, n = 12 per group). Data are shown as the means ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: P2rx1 knockout (KO) mice ( P2rx1 −/− ) constructed with the guidance of the CRISPR/Cas9 system were obtained from GemPharmatech Co. Ltd. (Jiangsu, China).

Techniques: Knock-Out, Expressing, Staining

Journal: Cell Biology and Toxicology

Article Title: P2rx1 deficiency alleviates acetaminophen-induced acute liver failure by regulating the STING signaling pathway

doi: 10.1007/s10565-023-09800-1

Figure Lengend Snippet: P2rx1 depletion eliminates APAP-induced cell death. (a) Representative images of TUNEL staining (original magnification × 200) and the statistical quantification of TUNEL-positive cells in liver sections of WT and P2rx1 −/− mice with or without APAP treatment (n = 4–6 per group); (b) Western blot and quantification analysis for the expression of hepatic BCL-2 and BCL-XL in WT and P2rx1 −/− mice with or without APAP treatment (n = 3–4); (c) Representative IHC staining (original magnification × 200) and the statistical quantification of hepatic cleaved-caspase-3-positive cells in liver sections of WT and P2rx1 −/− mice with or without APAP treatment (n = 4–6 per group); (d) Caspase-3 activity in the livers with or without APAP treatment (n = 4–6 per group); (e) Representative images of TUNEL staining (original magnification × 200, scale bar = 353 μm) and the quantification of TUNEL-positive primary hepatocytes with or without 5 mM APAP treatment in vitro (n = 3–6 per group). Data are shown as the means ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: P2rx1 knockout (KO) mice ( P2rx1 −/− ) constructed with the guidance of the CRISPR/Cas9 system were obtained from GemPharmatech Co. Ltd. (Jiangsu, China).

Techniques: TUNEL Assay, Staining, Western Blot, Expressing, Immunohistochemistry, Activity Assay, In Vitro

Journal: Cell Biology and Toxicology

Article Title: P2rx1 deficiency alleviates acetaminophen-induced acute liver failure by regulating the STING signaling pathway

doi: 10.1007/s10565-023-09800-1

Figure Lengend Snippet: P2rx1 depletion enhances inflammation resolution in response to APAP treatment. (a) Serum TNF-α, IL-6, and MCP-1 levels of both genotypes after PBS or APAP treatment (n = 4–6 per group); (b) Relative hepatic Tnf-α , Il-6 , and Mcp-1 mRNA in both mice after PBS or APAP treatment (n = 4–6 per group); Representative IHC images and the quantification of (c) CD11b-positive cells and (d) MPO-positive cells in liver sections after PBS or APAP treatment (original magnification × 200, n = 4–6 per group). Data are shown as the means ± SEM, *p < 0.05, **p < 0.01

Article Snippet: P2rx1 knockout (KO) mice ( P2rx1 −/− ) constructed with the guidance of the CRISPR/Cas9 system were obtained from GemPharmatech Co. Ltd. (Jiangsu, China).

Techniques:

Journal: Cell Biology and Toxicology

Article Title: P2rx1 deficiency alleviates acetaminophen-induced acute liver failure by regulating the STING signaling pathway

doi: 10.1007/s10565-023-09800-1

Figure Lengend Snippet: P2rx1 depletion improves mitochondria dysfunction and STING signaling-mediated inflammation. (a) RNA-seq analysis of livers from mice 6 h after APAP treatment. Bubble chart showing the top 10 of upregulated and downregulated KEGG enrichment of the significant genes; (b) Enrichment plots from the GSEA analysis; (c) Hepatic MDA and plasma mtDNA levels in both genotypes after PBS or APAP treatment (n = 4–6 per group); (d) JC-1 analysis for mitochondrial membrane potentials in primary hepatocytes after PBS or 5 mM APAP treatment (origin magnification × 100, scale bar = 353 μm); (e) Representative images of MitoSOX Red probe in primary hepatocytes after PBS or 5 mM APAP treatment (origin magnification × 100, scale bar = 353 μm); (f) Western blot analysis for expression of the STING-TBK1-P65 signaling pathway in liver extracts of mice after PBS or APAP treatment. Data are shown as the means ± SEM, *p < 0.05, **p < 0.01

Article Snippet: P2rx1 knockout (KO) mice ( P2rx1 −/− ) constructed with the guidance of the CRISPR/Cas9 system were obtained from GemPharmatech Co. Ltd. (Jiangsu, China).

Techniques: RNA Sequencing, Clinical Proteomics, Membrane, Western Blot, Expressing

Journal: Cell Biology and Toxicology

Article Title: P2rx1 deficiency alleviates acetaminophen-induced acute liver failure by regulating the STING signaling pathway

doi: 10.1007/s10565-023-09800-1

Figure Lengend Snippet: STING activator aggravates liver injury of P2rx1 −/− mice after APAP treatment. (a) A schematic of STING activation by DMX (10 mg/kg) in the APAP overdose model. (b) Serum levels of ALT and AST in P2rx1 −/− mice with or without DMX pretreatment (10 mg/kg, n = 4–6 per group); (c) Representative images of H&E staining (original magnification × 100) and quantification of hepatic necrosis area in P2rx1 −/− mice with or without DMX pretreatment (n = 4–6 per group); (d) Representative images and the quantification of TUNEL-positive cells in liver sections of P2rx1 −/− mice with or without DMX pretreatment (original magnification × 200, n = 4–6 per group); (e) Hepatic MDA and (f) plasma mtDNA levels of P2rx1 −/− mice with or without DMX pretreatment; (g) Representative images of MitoSOX Red probe in primary P2rx1 −/− hepatocytes pretreated with DMSO or DMX. Data are shown as the means ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001

Article Snippet: P2rx1 knockout (KO) mice ( P2rx1 −/− ) constructed with the guidance of the CRISPR/Cas9 system were obtained from GemPharmatech Co. Ltd. (Jiangsu, China).

Techniques: Activation Assay, Staining, TUNEL Assay, Clinical Proteomics

Journal: Cell Biology and Toxicology

Article Title: P2rx1 deficiency alleviates acetaminophen-induced acute liver failure by regulating the STING signaling pathway

doi: 10.1007/s10565-023-09800-1

Figure Lengend Snippet: STING activator promotes inflammatory response induced by APAP treatment in P2rx1 −/− mice. Representative IHC images and qualification of (a) CD11b-positive cells and (b) MPO-positive cells in the liver tissues of P2rx1 −/− mice with or without DMX pretreatment (origin magnification × 200, n = 4–6 per group); (c) Serum TNF-α, IL-6, and MCP-1 levels of P2rx1 −/− mice with or without DMX pretreatment (n = 4–6 per group); (d) Relative hepatic Tnf-α , Il-6 , and Mcp-1 mRNA levels of P2rx1 −/− mice 6 h with or without DMX pretreatment (n = 4–6 per group); (e) Western blot for the expression of the STING-TBK1-P65 signaling pathway in liver tissues of P2rx1 −/− mice with or without DMX pretreatment. Data are shown as the means ± SEM, **p < 0.01, ***p < 0.001

Article Snippet: P2rx1 knockout (KO) mice ( P2rx1 −/− ) constructed with the guidance of the CRISPR/Cas9 system were obtained from GemPharmatech Co. Ltd. (Jiangsu, China).

Techniques: Western Blot, Expressing

Journal: Frontiers in Immunology

Article Title: P2RX1-Involved Glycolytic Metabolism Supports Neutrophil Activation in Acute Pancreatitis

doi: 10.3389/fimmu.2020.549179

Figure Lengend Snippet: Targeting purinergic receptor P2RX1 alleviated inflammatory responses in acute pancreatitis. (A) Caerulein (50 μg/kg) was intraperitoneally injected seven times hourly to induce acute pancreatitis in WT and P2RX1-KO mice. NF449 or vehicle was intraperitoneally injected immediately after the first injection of caerulein. Saline-treated mice were set as negative control (NC). Pancreas tissues from each group were harvested at 8 h, stained with H&E, and observed under a microscope (100×). Representative images were shown. Scale bar is 50 μm. (B) At 8 h after caerulein exposure, serum amylase activity, pancreas IL1- β mRNA expression, Cxcl1 mRNA expression, myeloperoxidase (MPO) activity, and malondialdehyde (MDA) were analyzed to evaluate inflammatory status (n = 5 per group). 2 − δδ Ct value was used for comparisons of the fold change of mRNA expression to NC mice. Statistics were calculated using one-way ANOVA with Tukey post-tests. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: With the help from GemPharmatech Co., Ltd., global P2RX1 knockout (KO) mice were constructed in the C57BL/6 background with CRISPR/Cas9 system.

Techniques: Injection, Saline, Negative Control, Staining, Microscopy, Activity Assay, Expressing

Journal: Frontiers in Immunology

Article Title: P2RX1-Involved Glycolytic Metabolism Supports Neutrophil Activation in Acute Pancreatitis

doi: 10.3389/fimmu.2020.549179

Figure Lengend Snippet: Neutrophil-expressed P2RX1 contributes to the progression of acute pancreatitis. (A) Caerulein (50 μg/kg) was intraperitoneally injected seven times hourly to induce acute pancreatitis in bone marrow chimeras (WT → WT, KO → KO, WT → KO, and KO → WT). At 8 h after caerulein exposure, serum amylase activity, pancreas IL1- β , and pancreas Cxcl1 expression were analyzed to evaluate inflammatory status (n = 4 per group). (B) Caerulein (50 μg/kg) was intraperitoneally injected seven times hourly to induce acute pancreatitis in WT and P2RX1-KO mice. Saline-treated mice were set as negative control (NC). At 8 h after caerulein exposure, flow cytometry was performed to detected CD62L and CD11b expression in blood neutrophils (n = 5 each group). (C) Neutrophils in WT mice were depleted by intraperitoneal injection of anti-Ly6G antibody (1A8) 24 h before the first injection of caerulein. Bone marrow neutrophils isolated from of WT or P2RX1-KO mice were intravenously injected 1 h before the first administration of caerulein. At 8 h after caerulein exposure, serum amylase activity, pancreas IL1- β , and pancreas Cxcl1 mRNA expressions were analyzed to evaluate inflammatory status (n = 4 per group). 2 − δδ Ct value was used for comparisons of the fold change of mRNA expression to NC mice. Statistics were calculated using one-way ANOVA with Tukey post-tests (A, C) , or Student’s t test (B) . * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: With the help from GemPharmatech Co., Ltd., global P2RX1 knockout (KO) mice were constructed in the C57BL/6 background with CRISPR/Cas9 system.

Techniques: Injection, Activity Assay, Expressing, Saline, Negative Control, Flow Cytometry, Isolation

Journal: Frontiers in Immunology

Article Title: P2RX1-Involved Glycolytic Metabolism Supports Neutrophil Activation in Acute Pancreatitis

doi: 10.3389/fimmu.2020.549179

Figure Lengend Snippet: P2RX1 is required for facilitating neutrophil’ activation and glycolytic metabolism in acute pancreatitis. (A) Neutrophils were isolated from pancreas of WT or P2RX1-KO mice at 8 h after the first administration of caerulein. Expressions of inflammatory cytokines and glycolytic metabolism genes were detected by RT-qPCR. 2 − δδ Ct value was used for comparisons the fold change of mRNA expression to WT neutrophils. (B) Neutrophils were isolated from bone marrow of unstimulated WT or P2RX1-KO mice. Serum was used to stimulated neutrophils in the presence or absence of 2-DG for 6 h. Inflammatory cytokines were measured by RT-qPCR. 2 − δδ Ct value was used for comparisons the fold change of mRNA expression to vehicle serum-treated WT neutrophils. (C) Neutrophils were isolated from bone marrow of WT or P2RX1-KO mice. After stimulation with AP mice serum for 6 h, extracellular acid ratio (ECAR) was measured. (D) Pancreas P2RX1 expression in NC mice or AP mice were determined by RT-qPCR. 2 − δδ Ct value was used for comparisons of the fold change of mRNA expression to NC pancreas. (E) Neutrophils were isolated from bone marrow of WT or P2RX1-KO mice and stimulated with AP serum. Extracellular ATP was detected in indicated time points. (F) Neutrophils were isolated from bone marrow of WT or P2RX1-KO mice and stimulated with AP serum with or without apyrase. Inflammatory cytokines were measured by RT-qPCR. 2 − δδ Ct value was used for comparisons of the fold change of mRNA expression. Statistics were calculated using Student’s t test (A, E, F) , or one-way ANOVA with Tukey post-tests (B, C) . * P < 0.05, ** P < 0.01, *** p < 0.001.

Article Snippet: With the help from GemPharmatech Co., Ltd., global P2RX1 knockout (KO) mice were constructed in the C57BL/6 background with CRISPR/Cas9 system.

Techniques: Activation Assay, Isolation, Quantitative RT-PCR, Expressing

Journal: Nature Communications

Article Title: Identification of a subset of immunosuppressive P2RX1-negative neutrophils in pancreatic cancer liver metastasis

doi: 10.1038/s41467-020-20447-y

Figure Lengend Snippet: a Volcano plots of differential gene expression in 145 primary PDAC, 46 adjacent pancreases, 25 liver metastases and 27 adjacent livers. Red dots represent upregulated immune-related genes, and blue dots represent downregulated immune-related genes. b Immunome analyses of 26 infiltrating immune cell types in adjacent pancreas, primary PDAC, adjacent liver tissue and metastatic PDAC samples. c GO Biological Process analyses of differentially expressed genes between adjacent liver tissue and metastatic PDAC samples. d Expression analyses of P2RX1 in the adjacent pancreas, primary PDAC, adjacent liver tissue and metastatic PDAC samples from the GSE71729 and Renji cohorts. e Correlation analyses between P2RX1 and immune checkpoint molecules in metastatic PDAC samples. Bars represent mean ± standard deviation in ( d ). * P < 0.05, ** P < 0.01, and *** P < 0.001, by one-way ANOVA and Tukey’s multiple comparisons test ( d left), or Student’s t test ( d right). Source data are provided as a Source data file.

Article Snippet: With the help from GemPharmatech Co., Ltd., P2RX1 knockout ( P2rx1 −/− ) mice were constructed.

Techniques: Gene Expression, Expressing, Standard Deviation

Journal: Nature Communications

Article Title: Identification of a subset of immunosuppressive P2RX1-negative neutrophils in pancreatic cancer liver metastasis

doi: 10.1038/s41467-020-20447-y

Figure Lengend Snippet: a , b P2rx1 −/− mice were generated using CRISPR/Cas9 system. Schematic diagram was shown in ( a ) and genotyping results were shown in ( b ) (representative result from three independent experiments). c , d KPC cells were intrasplenically injected to seed livers of WT and P2rx1 −/− mice, and in vivo imaging was performed at sequential times. Representative images are shown in ( c ), and quantitative results are shown in ( d ) ( n = 6 per group, three independent experiments). e Representative images of liver metastatic samples harvested at day 17. f Liver weight of liver metastatic samples was measured at day 17 ( n = 5 per group, two independent experiments). g Survival analysis of liver metastatic WT or P2rx1 −/− mice within a duration of 5 weeks ( n = 10 per group, two independent experiments). h Normal liver (D0) and two sequential stages (D3 and D17) of liver metastases in WT and P2rx1 −/− mice were harvested for RNA-seq, and PCA analyses were performed ( n = 3 for D0, n = 4 for D3 and D17). i Representative Ki67 immunohistochemical staining of WT and P2rx1 −/− liver metastases at day 17 ( n = 4 per group, two independent experiments). 100 μm of scale bar for low power fields, 25 μm of scale bar for high power fields. j Heatmap of immune checkpoint molecules in liver metastases of WT or P2rx1 −/− mice at D3 and D17 ( n = 4 per group). Bars represent mean ± standard deviation in ( d , f ). P values are derived from two-sided Student’s t test ( d , f ), or log-rank test ( g ). Source data are provided as a Source data file.

Article Snippet: With the help from GemPharmatech Co., Ltd., P2RX1 knockout ( P2rx1 −/− ) mice were constructed.

Techniques: Generated, CRISPR, Injection, In Vivo Imaging, RNA Sequencing, Immunohistochemical staining, Staining, Standard Deviation, Derivative Assay

Journal: Nature Communications

Article Title: Identification of a subset of immunosuppressive P2RX1-negative neutrophils in pancreatic cancer liver metastasis

doi: 10.1038/s41467-020-20447-y

Figure Lengend Snippet: a Single cell suspension was obtained from mouse spleen and expression of P2RX1 was determined in indicated immune cell types ( n = 3 per group, two independent experiments). Left dotted line indicates the mean of negative control (NC) (secondary antibody only), and right dotted line indicates the mean of Ly6G+ cells. b KPC cells were intrasplenically injected to seed livers of WT mice. A single cell suspension was obtained from liver metastases and adjacent liver tissues of WT mice at day 17. The frequency of CD45+P2RX1+ cells was determined by flow cytometry ( n = 4 per group, three independent experiments). c KPC cells were intrasplenically injected to seed livers of WT mice. Immune cells were enriched from single cell suspension of liver metastases and adjacent liver tissues at day 17. P2RX1 expression in the indicated immune cell types was determined by flow cytometry ( n = 4 per group, three independent experiments). d Representative images of H&E staining and immunofluorescence staining of P2RX1 (Green), Ly6G (Red) and DAPI (Blue) in KPC mice spontaneous liver metastases (representative results from six independent experiments). 50 μm scale bar for low power fields and 20 μm scale bar for high power fields. e , f H&E staining and immunofluorescence staining of P2RX1 (green), CD66b (red) and DAPI (blue) in a total of 20 clinical PDAC liver metastasis samples were performed. Representative images are shown in ( e ), and the percentages of P2RX1-CD66b+ cells are shown in ( f ). 50 μm scale bar for low power fields and 20 μm scale bar for high power fields. g , h KPC cells were intrasplenically injected to seed livers of BM chimeras: WT → WT, P2rx1 −/− → P2rx1 −/− , WT → P2rx1 −/− , and P2rx1 −/− → WT. Neutrophils were depleted in WT → P2rx1 −/− and P2rx1 −/− → WT mice by intraperitoneal injection of anti-Ly6G (clone 1A8) antibody. At day 17, liver metastases were analyzed by in vivo imaging ( g , n = 5 for WT → WT, P2rx1 −/− → P2rx1 −/− and WT → P2rx1 −/− groups, n = 4 for WT → P2rx1 −/− + anti-Ly6G, P2rx1 −/− → WT and P2rx1 −/− → WT + anti-Ly6G groups, two independent experiments), and representative images of liver metastatic samples were shown ( h ). Bars represent mean ± standard deviation in ( f , g ). P values are derived from two-sided Student’s t test ( c , f ), or one-way ANOVA and Tukey’s multiple comparisons test ( g ). Source data are provided as a Source data file.

Article Snippet: With the help from GemPharmatech Co., Ltd., P2RX1 knockout ( P2rx1 −/− ) mice were constructed.

Techniques: Suspension, Expressing, Negative Control, Injection, Flow Cytometry, Staining, Immunofluorescence, In Vivo Imaging, Standard Deviation, Derivative Assay

Journal: Nature Communications

Article Title: Identification of a subset of immunosuppressive P2RX1-negative neutrophils in pancreatic cancer liver metastasis

doi: 10.1038/s41467-020-20447-y

Figure Lengend Snippet: a – g KPC cell was intrasplenically injected to seed livers of WT mice. Bone marrow (BM) and peripheral blood (PB) were obtained at day 17. Frequencies of neutrophils, P2RX1− neutrophils, and CXCR4+ neutrophils were determined by flow cytometry and quantitative results were shown ( n = 4 per group, three independent experiments). h Bone marrow neutrophils were isolated from WT mice and stimulated with indicated stimulus. RNA-seq were performed and Log 2 (FPKM+0.001) value of purinergic receptors were shown ( n = 1 per group). Bars represent mean ± standard deviation in ( e – g ). P values are derived from two-sided Student’s t test ( e – g ). Source data are provided as a Source data file.

Article Snippet: With the help from GemPharmatech Co., Ltd., P2RX1 knockout ( P2rx1 −/− ) mice were constructed.

Techniques: Injection, Flow Cytometry, Isolation, RNA Sequencing, Standard Deviation, Derivative Assay

Journal: Nature Communications

Article Title: Identification of a subset of immunosuppressive P2RX1-negative neutrophils in pancreatic cancer liver metastasis

doi: 10.1038/s41467-020-20447-y

Figure Lengend Snippet: a , b KPC cells were intrasplenically injected to seed livers of WT and P2rx1 −/− mice. A single cell suspension was obtained from liver metastases at day 17. Then, P2RX1+ neutrophils were purified from WT mice, and P2rx1 −/− neutrophils were purified from P2rx1 −/− mice for RNA sequencing. The results of KEGG analysis are shown in ( a ), and comparative expression of genes is shown in ( b ) ( n = 1 per group). c Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with tumor conditioned medium (TCM). The ECAR and OCR were then measured by a Seahorse assay in ( c ), and PD-L1 and TNF-α were detected by RT-qPCR in ( d ) ( n = 4 per group, two independent experiments). Glc, glucose; O (ECAR), oligomycin; 2-DG, 2-deoxyglucose; O (OCR), oligomycin; F, FCCP (carbonyl cyanide 4-[trifluoromethoxy] phenylhydrazone); A & R, antimycin A and rotenone. Bars represent mean ± standard deviation in ( c , d ). P values are derived from two-sided Student’s t test ( d ). Source data are provided as a Source data file.

Article Snippet: With the help from GemPharmatech Co., Ltd., P2RX1 knockout ( P2rx1 −/− ) mice were constructed.

Techniques: Injection, Suspension, Purification, RNA Sequencing, Expressing, Isolation, Quantitative RT-PCR, Standard Deviation, Derivative Assay

Journal: Nature Communications

Article Title: Identification of a subset of immunosuppressive P2RX1-negative neutrophils in pancreatic cancer liver metastasis

doi: 10.1038/s41467-020-20447-y

Figure Lengend Snippet: a Gene set enrichment analysis comparing RNA-seq data of P2RX1+ neutrophils and P2rx1 −/− neutrophils based on Nrf2 target genes. The p value and normalized enrichment score (NES) were shown. b , c Single-cell suspensions were obtained from liver metastases of WT and P2rx1 −/− mice at day 17. Intracellular Nrf2 was stained and detected by flow cytometry ( b ) or laser scanning confocal microscopy ( c ) in WT P2RX1+ neutrophils and P2rx1 −/− neutrophils ( n = 4 per group, three independent experiments). The scale bar is 2.5 μm. d Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with LPS + IFN-γ in the presence or absence of a Nrf2 inhibitor. The ECAR was then measured by a Seahorse assay ( n = 4 per group, two independent experiments). Glc, glucose; O, oligomycin; 2-DG, 2-deoxyglucose. e Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with IL-4 in the presence or absence of a Nrf2 inhibitor. The OCR was then measured by a Seahorse assay ( n = 4 per group, two independent experiments). O, oligomycin; F, FCCP (carbonyl cyanide 4-[trifluoromethoxy] phenylhydrazone); A & R, antimycin A and rotenone. f Bone marrow neutrophils were isolated from WT or P2rx1 −/− mice and stimulated with LPS + IFN-γ and an inhibitor Nrf2 inhibitor. IL-1β and TNF-α were determined by RT-qPCR ( n = 4 per group, three independent experiments). Bars represent mean ± standard deviation in ( d – f ). P values are derived from permutation test ( a ), two-sided Student’s t test ( f ). Source data are provided as a Source data file.

Article Snippet: With the help from GemPharmatech Co., Ltd., P2RX1 knockout ( P2rx1 −/− ) mice were constructed.

Techniques: RNA Sequencing, Staining, Flow Cytometry, Confocal Microscopy, Isolation, Quantitative RT-PCR, Standard Deviation, Derivative Assay

Journal: Nature Communications

Article Title: Identification of a subset of immunosuppressive P2RX1-negative neutrophils in pancreatic cancer liver metastasis

doi: 10.1038/s41467-020-20447-y

Figure Lengend Snippet: a A single cell suspension was obtained from liver metastases of WT and P2rx1 −/− mice at day 17. Flow cytometry was performed to detect the frequency of CD8+PD-1+ (upper) and Ly6G+PD-L1 + or Ly6G+PD-L1− (lower) cells. P2RX1 expression was further determined in Ly6G+PD-L1+ and Ly6G+PD-L1− cells from WT mice ( n = 4 per group, three independent experiments). b Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with tumor conditioned medium (TCM) or GM-CSF in the presence of a Nrf2 inhibitor or anti-GM-CSF neutralizing antibody. PD-L1 expression was detected by flow cytometry ( n = 4 per group, three independent experiments). c Integrative Genomics Viewer (IGV) was used to predict the two peaks that PD-L1 gene might be mediated by Nrf2 (upper). Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with GM-CSF. Binding of PD-L1 gene by Nrf2 was detected by ChIP-PCR ( n = 3 per group, two independent experiments). d Bone marrow neutrophils were isolated from WT and P2rx1 −/− mice and stimulated with GM-CSF. Intracellular ROS was detected by flow cytometry ( n = 4 per group, three independent experiments). e Antigen activated CTLs was co-cultured with GM-CSF primed WT or P2rx1 −/− neutrophils. Cell proliferation was analyzed with CSFE staining in the presence or absence of anti-PD-1 neutralizing antibody ( n = 4 per group, three independent experiments). f KPC cells were transfected with empty lentiviral vector (KPC-LV) or OVA (KPC-OVA). Antigen-activated CTLs were co-cultured with KPC-LV or KPC-OVA cells in the presence of GM-CSF-primed WT or P2rx1 −/− neutrophils. Cytotoxicity was determined in the presence or absence of anti-PD-1 neutralizing antibody by counting the number of PI+ cells ( n = 4 per group, three independent experiments). Bars represent mean ± standard deviation in ( f ). P values are derived from one-way ANOVA and Tukey’s multiple comparisons test ( f ). Source data are provided as a Source data file.

Article Snippet: With the help from GemPharmatech Co., Ltd., P2RX1 knockout ( P2rx1 −/− ) mice were constructed.

Techniques: Suspension, Flow Cytometry, Expressing, Isolation, Binding Assay, Cell Culture, Staining, Transfection, Plasmid Preparation, Standard Deviation, Derivative Assay