Journal: The Journal of Headache and Pain

Article Title: Src family kinases activity is required for transmitting purinergic P2X7 receptor signaling in cortical spreading depression and neuroinflammation

doi: 10.1186/s10194-021-01359-8

Figure Lengend Snippet: Both deactivation of P2X7 receptor and SFKs reduced cortical susceptibility to CSD in mouse brain slices. a The captured images of a mouse brain slice before and after CSD induction by 260 mM KCl in cerebral cortex. The arrow indicated the direction of CSD propagation. An AOI was selected and kept the same for data analysis. b The biphasic CSD curve generated from the images recorded for 15 minutes by plotting averaged grey level within the AOI against time. CSD latency (sec) is the time interval between KCl application and CSD elicitation at the AOI. CSD propagation rate (mm/min) is the velocity by which CSD propagates along cerebral cortex. c Representative traces of the 1st peak of CSD affected by 0.03% DMSO, 3 μM A740003 and 0.5 μM saracatinib. Only the trace recorded during the first 100 seconds was displayed here in order to clearly show the starting points of KCl application and CSD elicitation. d e Effects of 0.03% DMSO, 3 μM A740003 and 0.5 μM saracatinib on CSD latency and propagation rate. Abbreviations: saracatinib (SRCT); seconds (sec); mm/minute (mm/min). Two-tailed unpaired t-test was used for comparison in CSD latency and propagation rate between DMSO and A740003 group, DMSO and saracatinib group. Significant differences were indicated by * p < 0.05, ** p < 0.01

Article Snippet: Three series of studies were designed using this ex vivo CSD model. Series 1: In order to study whether modulation of SFKs or P2X7 receptor activity could similarly regulate cortical susceptibility to CSD, the effects of a SFKs inhibitor, saracatinib (S1006, Selleckchem, Houston, USA), that is currently investigated in clinical trials for treating several neurological disorders [ , ] or a P2X7 receptor antagonist, A740003 (3701, Tocris, Bristol, UK) on CSD latency and propagation rate were examined respectively in C57BL/6J mice brain slices.

Techniques: Slice Preparation, Generated, Two Tailed Test, Comparison

Journal: The Journal of Headache and Pain

Article Title: Src family kinases activity is required for transmitting purinergic P2X7 receptor signaling in cortical spreading depression and neuroinflammation

doi: 10.1186/s10194-021-01359-8

Figure Lengend Snippet: Disrupting P2X7 receptor-SFKs interaction reduced cortical susceptibility to CSD and CSD-associated neuroinflammation in mouse brain slices. a Representative traces of 1 st peak of CSD affected by Kreb’s, 3 μM TAT-P2X7 and TAT-P2X7SC. Only the trace recorded during the first 100 seconds was displayed here in order to clearly show CSD latency in respective group. b c Effects of Kreb’s, 3 μM TAT-P2X7 and TAT-P2X7SC on CSD latency and propagation rate. d e Effects of 3 μM TAT-P2X7 and TAT-P2X7SC on IL-1β and TNFα mRNA fold change after CSD in mouse brain slices. Two-tailed unpaired t-test was used for comparison in CSD latency and propagation rate between Kreb’s and TAT-P2X7 group, TAT-P2X7 and TAT-P2X7SC group; in IL-1β and TNFα mRNA fold change between TAT-P2X7 and TAT-P2X7SC group. Significant differences were indicated by * p < 0.05, ** p < 0.01

Article Snippet: Three series of studies were designed using this ex vivo CSD model. Series 1: In order to study whether modulation of SFKs or P2X7 receptor activity could similarly regulate cortical susceptibility to CSD, the effects of a SFKs inhibitor, saracatinib (S1006, Selleckchem, Houston, USA), that is currently investigated in clinical trials for treating several neurological disorders [ , ] or a P2X7 receptor antagonist, A740003 (3701, Tocris, Bristol, UK) on CSD latency and propagation rate were examined respectively in C57BL/6J mice brain slices.

Techniques: Two Tailed Test, Comparison

Journal: The Journal of Headache and Pain

Article Title: Src family kinases activity is required for transmitting purinergic P2X7 receptor signaling in cortical spreading depression and neuroinflammation

doi: 10.1186/s10194-021-01359-8

Figure Lengend Snippet: NMDA restored the disrupted P2X7 receptor-SFKs interaction-reduced cortical susceptibility to CSD but not CSD-associated neuroinflammation in mouse brain slices. a Effects of 3 μM TAT-P2X7 and TAT-P2X7SC on glutamate release from mouse brain slices after CSD. b Representative traces of 1 st peak of CSD affected by 3 μM TAT-P2X7 and 3 μM TAT-P2X7 + 10 μM NMDA. c d Effects of 3 μM TAT-P2X7 + 10 μM NMDA on CSD latency and propagation rate. e Effects of 3 μM TAT-P2X7 + 10 μM NMDA on CSD-associated IL-1β mRNA fold change in mouse brain slices. Two-tailed unpaired t-test was used for comparison in glutamate release between TAT-P2X7 and TAT-P2X7SC group; in CSD latency, propagation rate and IL-1β mRNA fold change between TAT-P2X7 and TAT-P2X7 + NMDA group. Significant differences were indicated by * p < 0.05

Article Snippet: Three series of studies were designed using this ex vivo CSD model. Series 1: In order to study whether modulation of SFKs or P2X7 receptor activity could similarly regulate cortical susceptibility to CSD, the effects of a SFKs inhibitor, saracatinib (S1006, Selleckchem, Houston, USA), that is currently investigated in clinical trials for treating several neurological disorders [ , ] or a P2X7 receptor antagonist, A740003 (3701, Tocris, Bristol, UK) on CSD latency and propagation rate were examined respectively in C57BL/6J mice brain slices.

Techniques: Two Tailed Test, Comparison

Journal: The Journal of Headache and Pain

Article Title: Src family kinases activity is required for transmitting purinergic P2X7 receptor signaling in cortical spreading depression and neuroinflammation

doi: 10.1186/s10194-021-01359-8

Figure Lengend Snippet: Schematic representation of the role that P2X7 receptor/SFKs signaling may exert in CSD-associated migraine pathophysiology. P2X7 receptor/SFKs signaling is activated during CSD to facilitate neuroinflammation, NMDA receptor activation and glutamate release. Glutamate may in return reinforce the activation of NMDA receptor (dotted line with arrow), both of which facilitate cortical susceptibility to CSD, forming a positive loop

Article Snippet: Three series of studies were designed using this ex vivo CSD model. Series 1: In order to study whether modulation of SFKs or P2X7 receptor activity could similarly regulate cortical susceptibility to CSD, the effects of a SFKs inhibitor, saracatinib (S1006, Selleckchem, Houston, USA), that is currently investigated in clinical trials for treating several neurological disorders [ , ] or a P2X7 receptor antagonist, A740003 (3701, Tocris, Bristol, UK) on CSD latency and propagation rate were examined respectively in C57BL/6J mice brain slices.

Techniques: Activation Assay

Journal: Neurobiology of disease

Article Title: P2RX7 modulates the function of human microglia-like cells and mediates the association of IL18 with Alzheimer’s disease traits

doi: 10.1016/j.nbd.2025.107106

Figure Lengend Snippet: Extracellular ATP induces IL1β and IL18 gene expression upregulation in a P2RX7-dependent manner. A) IL1β , IL18 , and NLRP3 expression levels were measured with qPCR from MDMi treated with DMSO alone (Ctrl), 1 mM ATP (ATP), or 1 mM ATP plus 10 μM A740003 (ATP + A74). Statistics were determined with a repeated measure one-way paired ANOVA with Newman-Keuls multiple comparisons test. N = 14. B) IL-1β ( N = 6) and IL-18 ( N = 5) protein detection in supernatants of MDMi treated with DMSO alone (Ctrl), 1 mM ATP (ATP), or 1 mM ATP plus 10 μM A740003 (ATP + A74). Statistics were determined with a two-way paired ANOVA. C) IL1β , IL18 , and NLRP3 expression levels were measured with qPCR from MDMi treated with DMSO alone (Ctrl), or 500 μM bzATP (bzATP). Statistics were determined with a paired t -test. N = 9. ns = p > 0.05, * p ≤ 0.05, ** ≤ 0.01,*** ≤ 0.001, **** ≤ 0.0001.

Article Snippet: The A740003 P2RX7 antagonist (Tocris biotechne, #861393–28–4, Batch #3B/266155) was dissolved in DMSO and added to MDMi cultures at a concentration of 10 μM.

Techniques: Gene Expression, Expressing

Journal: Neurobiology of disease

Article Title: P2RX7 modulates the function of human microglia-like cells and mediates the association of IL18 with Alzheimer’s disease traits

doi: 10.1016/j.nbd.2025.107106

Figure Lengend Snippet: ATP-mediated P2RX7 activation reduces MDMi Aβ1–42 uptake abilities. A) Mean fluorescence intensity (MFI) of HyLite Fluor 647-labeled Aβ1–42 peptide uptake normalized to DAPI MFI in MDMi treated with DMSO alone (Ctrl), 1 mM ATP (ATP), or 1 mM ATP and 10 μM A740003 (ATP + A74), measured by fluorescence on a microplate reader. Data are represented relative to the control condition. Statistics were determined with a repeated measure one-way paired ANOVA with Newman-Keuls multiple comparisons test. N = 26. B) MFI of HyLite Fluor 647-labeled Aβ1–42 peptide uptake normalized to DAPI MFI in MDMi treated with DMSO alone (Ctrl), or 500 μM bzATP (bzATP), as measured by fluorescence on a microplate reader. Data are represented relative to the control condition. Statistics were determined with a paired t-test. N = 14. C) Mean fluorescence intensity (MFI) of HyLite Fluor 647-labeled Aβ1–42 per DAPI+ cell in MDMi imaged using confocal microscopy. MDMi were treated with DMSO alone (Ctrl), 1 mM ATP (ATP), or 1 mM ATP and 10 μM A740003 (ATP + A74). Statistics were determined with a repeated measure one-way paired ANOVA with Newman-Keuls multiple comparisons test. N = 12. D) Representative images of MDMi treated with DMSO alone (Ctrl), 1 mM ATP (ATP), or 1 mM ATP and 10 μM A740003 (ATP + A74) and stained for P2RX7 (green) and DAPI (blue) and used to measure uptake of HyLite Fluor 647-labeled Aβ1–42 peptide (red). Images were processed in CellProfiler. ns = p > 0.05, *p ≤ 0.05, ** ≤ 0.01,*** ≤ 0.001, **** ≤ 0.0001.

Article Snippet: The A740003 P2RX7 antagonist (Tocris biotechne, #861393–28–4, Batch #3B/266155) was dissolved in DMSO and added to MDMi cultures at a concentration of 10 μM.

Techniques: Activation Assay, Fluorescence, Labeling, Control, Confocal Microscopy, Staining

Journal: Neurobiology of disease

Article Title: P2RX7 modulates the function of human microglia-like cells and mediates the association of IL18 with Alzheimer’s disease traits

doi: 10.1016/j.nbd.2025.107106

Figure Lengend Snippet: IL18 but not IL1β is correlated with P2RX7 gene expression in MDMi. IL1β , IL18 , and P2RX7 expression levels were measured with qPCR from MDMi. A) Correlation of IL1β and P2RX7 expression. Pearson correlation coefficient (r) = −0.002, two-tailed P value = 0.99. B) Correlation of IL18 and P2RX7 expression. Pearson correlation coefficient (r) = 0.59, two-tailed P value = 0.02. R.E. = Relative Gene Expression (2^−(ΔCt)*1000) to GAPDH . N = 15.

Article Snippet: The A740003 P2RX7 antagonist (Tocris biotechne, #861393–28–4, Batch #3B/266155) was dissolved in DMSO and added to MDMi cultures at a concentration of 10 μM.

Techniques: Gene Expression, Expressing, Two Tailed Test

Journal: Oncogene

Article Title: The P2X7 receptor modulates immune cells infiltration, ectonucleotidases expression and extracellular ATP levels in the tumor microenvironment

doi: 10.1038/s41388-019-0684-y

Figure Lengend Snippet: P2X7 loss enriches tumor infiltrate in Tregs overexpressing OX40, PD-1, and CD73 and decreases proinflammatory cytokines. a–c C57bl/6 mice were inoculated into the right hind flank with B16-pmeLUC cells in WT and P2X7 null mice. a Tumor volume was in vivo assessed at the indicated time points, b ex vivo tumor volume assessed by a calliper, and c representative pictures of tumors from WT and P2X7 null mice at post-inoculum day 14. Data are shown as the mean ± SEM (WT, n = 6–12; P2X7 null, n = 5–12). d–l Flow cytometric analysis of tumor masses from C57bl/6 WT and P2X7 null mice. d Percentage of CD8 + infiltrating T cells, e percentage of Teff (CD25, Foxp3, CD4 + gate), f percentage of Tregs (CD25 + , Foxp3 + , CD4 + gate), g–i mean fluorescence intensity (MFI) of OX40 ( g ), PD-1( h ), CD73 ( i ) on Tregs, j MFI of CD73 on Teff, k MFI of CD73 on monocytes/macrophages gate (CD11b + Ly6C + F4/80 + ), l MFI of CD39 on CD4 + , CD25 − Teff. Data are shown as the mean ± SEM (WT, n = 4–9; P2X7 null, n = 4–9). m–p Levels of plasma cytokines of tumor-bearing C57/bl6 mice inoculated with B16-pmeLUC cells. TGF-β1 ( m ), IL-1β ( n ), IFN-γ ( o ), and TNF-α ( p ) were evaluated in plasma samples obtained at post-inoculum day 14. Data are shown as the mean ± SEM (WT, n = 5–14; P2X7 null, n = 5–11). * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: P2X7 antagonist A740003 (Tocris Bioscience, Bristol, UK) or vehicle (sterile phosphate-buffered saline (PBS), 0.005% DMSO) were intra-peritoneum injected (100 μl) every 2 days after day 5, corresponding, when applicable, to first tumor mass detection.

Techniques: In Vivo, Ex Vivo, Fluorescence, Clinical Proteomics

Journal: Oncogene

Article Title: The P2X7 receptor modulates immune cells infiltration, ectonucleotidases expression and extracellular ATP levels in the tumor microenvironment

doi: 10.1038/s41388-019-0684-y

Figure Lengend Snippet: P2X7 antagonism increases tumor-infiltrating T effector lymphocytes and decreases ectonucleotidases expression on T effector and dendritic cells. a–c C57bl/6 mice were inoculated into the right hind flank with B16-pmeLUC in WT mice. A740003 (50 µg/kg) was intra-peritoneum administered to mice at post-inoculum days 5, 7, 9, 11, and 13. a Tumor volume was in vivo assessed at the indicated time points, b ex vivo tumor volume assessed by a calliper, c representative pictures of tumors from treated mice at post-inoculum day 14. Data are shown as the mean ± SEM ( n = 9 per group). d–j Flow cytometric analysis of tumor masses from C57bl/6 WT mice treated with placebo or A740003 (50 µg/kg). d Percentage of CD4 +- infiltrating T cells (CD45 + T gate), e percentage of Teff (CD25 - , Foxp3, CD4 + gate), f mean fluorescence intensity (MFI) of OX40 on Tregs, g, h MFI of CD39 ( g ) and CD73 ( h ) on Teff, i, j MFI of CD39 ( i ) and CD73 ( j ) on cDC (cDC, Cd11b + Ly6C low , Cd11c + gate). Data are shown as the mean ± SEM. (placebo, n = 6–8; A740003, n = 4–8). k, l P2X7 antagonism increases circulating levels of IFN-γ while decreasing IL-1β. IFN-γ ( n = 6 per group) ( k ) and IL-1β ( l ) ( n = 12 per group) were evaluated in plasma samples obtained at post-inoculum day 14. Data are shown as the mean ± SEM. * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: P2X7 antagonist A740003 (Tocris Bioscience, Bristol, UK) or vehicle (sterile phosphate-buffered saline (PBS), 0.005% DMSO) were intra-peritoneum injected (100 μl) every 2 days after day 5, corresponding, when applicable, to first tumor mass detection.

Techniques: Expressing, In Vivo, Ex Vivo, Fluorescence, Clinical Proteomics

Journal: Oncogene

Article Title: The P2X7 receptor modulates immune cells infiltration, ectonucleotidases expression and extracellular ATP levels in the tumor microenvironment

doi: 10.1038/s41388-019-0684-y

Figure Lengend Snippet: P2X7 ablation leads to a decrease in tumor ATP levels. a–g C57bl/6 ( a, b ) and BALBc/J ( c–g ) mice were inoculated into the right hind flank with B16-pmeLUC or WEHI-3B-pmeLUC cells, respectively in WT and P2X7 null mice. a, f Measure of ATP levels in tumor-bearing mice estimated by pmeLUC luminescence emission (p/s/cm 2 /sr), b, g representative pictures of pmeLUC luminescence emission in C57bl/6 ( b ) tumor-bearing mice at post-inoculum days 5, 7, and 9 and in BALBc/J ( g ) tumor-bearing mice at post-inoculum day 7, c tumor volume was in vivo assessed at the indicated time points, d ex vivo tumor volume assessed by a calliper, e representative pictures of tumors from WT and P2X7 null mice at post-inoculum day 14. Data are shown as the mean ± SEM (C57bl/6 WT, n = 6–12; C57bl/6 P2X7 null, n = 5–12; BALBc/J n = 12 per group). h–k Levels of plasma cytokines of tumor-bearing BALBc/J mice inoculated with WEHI-3B-pmeLUC cells. TGF-β1 ( h ) ( n = 9 per group), IL-1β ( i ) ( n = 17 per group), TNF-α ( j ) ( n = 10 per group), and IFN-γ ( k ) (WT, n = 4; P2X7 null, n = 5) were evaluated in plasma samples obtained at post-inoculum day 14. Data are shown as the mean ± SEM. * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: P2X7 antagonist A740003 (Tocris Bioscience, Bristol, UK) or vehicle (sterile phosphate-buffered saline (PBS), 0.005% DMSO) were intra-peritoneum injected (100 μl) every 2 days after day 5, corresponding, when applicable, to first tumor mass detection.

Techniques: In Vivo, Ex Vivo, Clinical Proteomics

Journal: Oncogene

Article Title: The P2X7 receptor modulates immune cells infiltration, ectonucleotidases expression and extracellular ATP levels in the tumor microenvironment

doi: 10.1038/s41388-019-0684-y

Figure Lengend Snippet: P2X7 null immune cells, alone or in co-colture with cancer cells, show lowered ATP release. a Supernatant ATP assessed in B16 and peritoneal macrophages coltures from WT and P2X7 null mice treated with either vehicle (PBS + DMSO 0,1%) or A740003 20 µM. Data are shown as the mean ± SEM (Vehicle, n = 7; A740003, n = 8). b, c Peri-cellular ATP levels in B16-pmeLUC cells ( b ) or WEHI-3B-pmeLUC cells ( c ) alone and in co-culture with peritoneal macrophages derived from C57bl/6 (B16-pmeLUC n = 17; Co-culture WT n = 34; Co-culture P2X7 null n = 25) or BALBc/J (WEHI-3B-pmeLUC n = 6; Co-culture WT n = 6; Co-culture P2X7 null n = 9) WT and P2X7 null mice, respectively. Data are shown as the mean ± SEM. d Peri-cellular ATP levels in B16-pmeLUC cells alone or in co-culture with splenocytes derived from C57bl/6 WT and P2X7 null mice. Data are shown as the mean ± SEM (B16-pmeLUC n = 8; Co-culture WT n = 13; Co-culture P2X7 null n = 15). Luminescence data were expressed as total photons acquired in a 5 min session. * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: P2X7 antagonist A740003 (Tocris Bioscience, Bristol, UK) or vehicle (sterile phosphate-buffered saline (PBS), 0.005% DMSO) were intra-peritoneum injected (100 μl) every 2 days after day 5, corresponding, when applicable, to first tumor mass detection.

Techniques: Co-Culture Assay, Derivative Assay

Journal: Oncogene

Article Title: The P2X7 receptor modulates immune cells infiltration, ectonucleotidases expression and extracellular ATP levels in the tumor microenvironment

doi: 10.1038/s41388-019-0684-y

Figure Lengend Snippet: P2X7 antagonist systemic administration does not influence ATP levels in vivo. C57bl/6 ( a, b ) and BALBc/J ( c–g ) mice were inoculated into the right hind flank with B16-pmeLUC or WEHI-3B-pmeLUC cells, respectively, in WT mice. A740003 (50 µg/kg) was intra-peritoneum administered to mice at post-inoculum days 5, 7, 9, 11, and 13. a , f Measure of ATP levels in treated mice estimated by pmeLUC luminescence emission (p/s/cm 2 /sr), b , g representative picture of pmeLUC luminescence emission in mice at post-inoculum day 7, c tumor volume was in vivo assessed at the indicated time points, d ex vivo tumor volume assessed by a calliper, e representative pictures of tumors from treated mice at post-inoculum day 14. Data are shown as the mean ± SEM ( n = 9 per group). * P < 0.05 and ** P < 0.01

Article Snippet: P2X7 antagonist A740003 (Tocris Bioscience, Bristol, UK) or vehicle (sterile phosphate-buffered saline (PBS), 0.005% DMSO) were intra-peritoneum injected (100 μl) every 2 days after day 5, corresponding, when applicable, to first tumor mass detection.

Techniques: In Vivo, Ex Vivo

Journal: Oncogene

Article Title: The P2X7 receptor modulates immune cells infiltration, ectonucleotidases expression and extracellular ATP levels in the tumor microenvironment

doi: 10.1038/s41388-019-0684-y

Figure Lengend Snippet: A740003 treatment leads to B16-pmeLUC cell growth arrest accompanied by ATP release. a Proliferation at 0, 24, and 48 h of B16-pmeLUC cells treated with vehicle (PBS + 0.1% DMSO) or A740003 20 µM ( n = 6 per group). b ATP levels of B16-pmeLUC cells treated as above described. Luminescence data are normalized on cell number and expressed as total photons measured in a 5 min acquisition ( n = 6 per group). c, d P2X7 antagonism in co-culture of tumor cells and macrophages or splenocytes results in unaltered ATP levels. c ATP levels in B16-pmeLUC cells alone and in co-culture with peritoneal macrophages derived from C57bl/6 WT mice treated with vehicle (PBS + 0.1% DMSO) or A740003 20 µM (Vehicle, n = 4; A740003, n = 6), d ATP levels in B16-pmeLUC cells alone and in co-culture with splenocytes derived from C57bl/6 WT and P2X7 null mice treated with vehicle (PBS + 0.1% DMSO) or A740003 20 µM (Vehicle, n = 6–10; A740003, n = 9–12). e Tregs reduce ATP secretion from cancer cells. ATP levels in B16-pmeLUC cells alone and in co-culture with Treg cells derived from C57bl/6 WT and P2X7 null mice treated with vehicle (PBS + 0.1% DMSO) or A740003 20 µM ( n = 9 per group). c–e Luminescence data are expressed as total photons measured in a 5 min acquisition. Data are shown as the mean ± SEM. * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: P2X7 antagonist A740003 (Tocris Bioscience, Bristol, UK) or vehicle (sterile phosphate-buffered saline (PBS), 0.005% DMSO) were intra-peritoneum injected (100 μl) every 2 days after day 5, corresponding, when applicable, to first tumor mass detection.

Techniques: Co-Culture Assay, Derivative Assay

Journal: Oncogene

Article Title: The P2X7 receptor modulates immune cells infiltration, ectonucleotidases expression and extracellular ATP levels in the tumor microenvironment

doi: 10.1038/s41388-019-0684-y

Figure Lengend Snippet: P2X7 blockade reduces tumor growth in P2X7 null mice. a–e C57bl/6 mice were inoculated into the right hind flank with B16-pmeLUC in P2X7 null mice. A740003 (50 µg/kg) was intra-peritoneum administered to mice at post-inoculum days 5, 7, 9, 11, and 13. a Tumor volume was in vivo assessed at the indicated time points, b ex vivo tumor volume assessed by a calliper, c representative pictures of tumors from treated mice at post-inoculum day 14, d kinetics of ATP levels in treated mice estimated by pmeLUC luminescence emission (p/s/cm 2 /sr), e representative picture of pmeLUC luminescence emission in mice at post-inoculum day 9 (placebo, n = 7; A740003, n = 8). f–h Ex vivo characterization of T cells by cytometric analysis in tumor masses derived from C57bl/6 P2X7 null mice treated with placebo or A740003 (50 µg/kg). f Percentage of CD4 + infiltrating T cells ( n = 5 per group), g , h MFI of CD73 on Tregs ( g ) (placebo, n = 5; A740003, n = 7) and Teff ( h ) (placebo, n = 5; A740003, n = 6). Data are shown as the mean ± SEM. * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: P2X7 antagonist A740003 (Tocris Bioscience, Bristol, UK) or vehicle (sterile phosphate-buffered saline (PBS), 0.005% DMSO) were intra-peritoneum injected (100 μl) every 2 days after day 5, corresponding, when applicable, to first tumor mass detection.

Techniques: In Vivo, Ex Vivo, Derivative Assay

Journal: Oncogene

Article Title: The P2X7 receptor modulates immune cells infiltration, ectonucleotidases expression and extracellular ATP levels in the tumor microenvironment

doi: 10.1038/s41388-019-0684-y

Figure Lengend Snippet: Schematic representation of the main findings of the study. Tumors growing in P2X7 null host show an increase of volume accompanied by a generation of an immunosuppressive microenvironment, characterized by a decrease of ATP levels and an augmented number of regulatory T cells overexpressing OX40 and CD73. P2X7 pharmacological blockade leads to a reduction of tumor volume and an increase of tumor-infiltrating T lymphocytes that express low levels of CD39 and CD73. Tregs and conventional dendritic cells while remaining unaltered in numbers down-modulate, respectively, OX40, CD39 and CD73

Article Snippet: P2X7 antagonist A740003 (Tocris Bioscience, Bristol, UK) or vehicle (sterile phosphate-buffered saline (PBS), 0.005% DMSO) were intra-peritoneum injected (100 μl) every 2 days after day 5, corresponding, when applicable, to first tumor mass detection.

Techniques:

Journal: American Journal of Cancer Research

Article Title: CD39 + tumor infiltrating T cells from colorectal cancers exhibit dysfunctional phenotype

doi:

Figure Lengend Snippet: CD39 expression aggravates eATP-induced IL-2 suppression via generating adenosine. (A) Jurkat T cells were treated with indicated dose eATP with or without indicated activation cocktails for 24 hours. (B) Jurkat cells were pre-treated with P2X7 receptor inhibitors or A2A receptor for 1 hour before challenging with eATP and OKT3 for 24 hours. (C and D) Jurkat cells were pre-treated with CD39 inhibitors POM-1 and ARL67156 for 1 hour before adding eATP and OKT3. Cell-free supernatant was harvested for the detection of adenosine (C) and IL-2 (D). (E) Jurkat-PCDH-empty and Jurkat-PCDH-CD39 cells were treated with indicated concentration of OKT3 for 30 minutes. Cell lysates were prepared for WB. *P < 0.05, **P < 0.01, ***P < 0.001.

Article Snippet: P2X7 antagonists Brilliant Blue G (BBG) and A-740003 were purchased from Selleck Chemicals, Shanghai, China.

Techniques: Expressing, Activation Assay, Concentration Assay

Journal: Cell reports

Article Title: Single-cell transcriptomics of NRAS-mutated melanoma transitioning to drug resistance reveals P2RX7 as an indicator of early drug response.

doi: 10.1016/j.celrep.2023.112696

Figure Lengend Snippet: Figure 7. The impact of drug exposure on ROS production in NRASmut melanoma (A) ROS production after short-term (1 and 4 days) exposure to combined MEKi/CDK4/6i therapy in MelJuso (SAC) and SKMel30 (FAC) cells. Cell exposure to H2O2 for 30 min served as a positive control. Mean values ± SD from three biological replicates are plotted. (B) ROS production following P2RX7 silencing and/or activation with 100 mM BzATP in untreated or 4 days binimetinib/palbociclib-treated (B/P) MelJuso cells. RNAiMax, transfection reagent alone; NC, negative control siRNA. Bars indicate the mean values ± SD from three biological replicates. (C) Growth curves of SACs (MelJuso) during MEK/ CDK4/6 co-inhibition alone or with P2RX7 activation (100mMBzATP) and/orP2RX7antagonist A-740003 (1 mM), measured in an IncuCyte ZOOM live-cell microscope (representative results of mean of two technical replicates ±SEM). (D) ROS production after 4, 7, and 10 days of MEKi (binimetinib [B]) and CDK4/6i (palbociclib [P]) treat- ment (B/P) with or without BzATP (100 mM) and A-740003 (1 mM). For (A) and (B), statistical signifi- cance was established by one-way ANOVA, followed by multiple comparisons test; ns, not sig- nificant; *p % 0.05; **p % 0.01; ***p % 0.001; ****p % 0.0001.

Article Snippet: P2RX7 antagonists KN-62 and A-740003 were supplied from Selleck Chemicals.

Techniques: Positive Control, Activation Assay, Transfection, Negative Control, Inhibition, Microscopy