Journal: Journal of orthopaedic research : official publication of the Orthopaedic Research Society

Article Title: Potential Role for a Specialized β 3 Integrin-Based Structure On Osteocyte Processes in Bone Mechanosensation

doi: 10.1002/jor.23792

Figure Lengend Snippet: Antibodies Used in Co-Localization Studies

Article Snippet: After staining, sections were mounted in Eukitt mounting media (EM Sciences) on precision thickness 1.5 glass coverslips (ThermoFisher). table ft1 table-wrap mode="anchored" t5 caption a7 EXPERIMENT TARGET 1°ANTIBODY VENDOR 2°ANTIBODY VENDOR Pilot β 3 integrin #ab20146 Abcam # A-32723 AlexaFluor488 ThermoFisher β 3 integrin vs Vinculin β 3 integrin # ab20146 Abcam #A-11019 AlexaFluor568 ThermoFisher β 3 integrin vs Vinculin Vinculin #V9131 Sigma #A-11034 AlexaFluor488 ThermoFisher β 3 integrin vs Panx1,P2X7R,CaV3 β 3 integrin # ab75872 Abcam #A-10042 AlexaFluor568 ThermoFisher β 3 integrin vs Panx1 Panx1 #SC-49695 SantaCruz #A-11055 AlexaFluor488 ThermoFisher β 3 integrin vs P2X7R P2X7R # ab93354 Abcam #A-11055 AlexaFluor488 ThermoFisher β 3 integrin vs CaV3 Cav3 # SC-16263 SantaCruz #A-11055 AlexaFluor488 ThermoFisher β 3 integrin vs Cx43 Cx43 #C6219 Sigma #A-11034 AlexaFluor488 ThermoFisher Panx1 vs P2X7R, Panx1 #SC-49693 SantaCruz # {"type":"entrez-nucleotide","attrs":{"text":"A11055","term_id":"490909","term_text":"A11055"}} A11055 AlexaFluor488 ThermoFisher Panx1 vs CaV3 Panx1 #ab139715 Abcam #A-10042 AlexaFluor568 ThermoFisher Panx1 vs P2X7R P2X7R vs CaV3 P2X7R #ab109246 Abcam #A-10042 AlexaFluor568 ThermoFisher Panx1 vs CaV3 Cav3 #SC-16263 SantaCruz #A-11055 AlexaFluor488 ThermoFisher STORM FLAG F3165, mouse Sigma AlexaFluor488 Open in a separate window Antibodies Used in Co-Localization Studies SIM was performed using a Zeiss ELYRA S.1 system equipped with two laser channels; 1-2 μm z-axis image stacks were acquired in 110 nm optical steps (63× oil immersion objective, NA1.4).

Techniques:

Journal: Journal of orthopaedic research : official publication of the Orthopaedic Research Society

Article Title: Potential Role for a Specialized β 3 Integrin-Based Structure On Osteocyte Processes in Bone Mechanosensation

doi: 10.1002/jor.23792

Figure Lengend Snippet: (A) Wide-field fluorescence image of osteocyte double-IHC stained to detect β3 integrin (Red, AlexaFluor568) and Panx1 (Green, AlexaFluor488), (B) Corresponding thresholded SIM image of the same cell shown in a single 110 nm thick optical section (see Fig 1 for explanation of discontinuous appearance of cell processes in these very thin optical sections); (C) Negative control with non-immune species specific IgGs. (D) is the same image as in B, shown with concentric ellipses every 0.5 μm that were used in standardized sampling of staining foci on cell processes as a function of distance from the lacunar edge; the central gray oval represents the osteocyte lacunar edge and measurements were started 0.5 μm away from the edge. Note that this image has been rotated slightly counterclockwise compared to B, in order to place the osteocyte long axis in the horizontal plane. (E) Example of a composite stain locus, magnified (blue arrow) and shown in separate Red and Green component channels. Asterisks show the X, Y-location of the maximum intensity, which were used to determine distances. (F-H) show one optical plane of thresholded SIM images of β3 integrin (Red) vs P2X7R (Green), β3 integrin (Red) vs CaV3 (Green) and β3 integrin (Red) vs Cx43 (Green), respectively, and (I-K) negative controls (non-immune species specific IgGs) corresponding to the staining pair in the image shown immediately above. Scale bar = 5 μm.

Article Snippet: After staining, sections were mounted in Eukitt mounting media (EM Sciences) on precision thickness 1.5 glass coverslips (ThermoFisher). table ft1 table-wrap mode="anchored" t5 caption a7 EXPERIMENT TARGET 1°ANTIBODY VENDOR 2°ANTIBODY VENDOR Pilot β 3 integrin #ab20146 Abcam # A-32723 AlexaFluor488 ThermoFisher β 3 integrin vs Vinculin β 3 integrin # ab20146 Abcam #A-11019 AlexaFluor568 ThermoFisher β 3 integrin vs Vinculin Vinculin #V9131 Sigma #A-11034 AlexaFluor488 ThermoFisher β 3 integrin vs Panx1,P2X7R,CaV3 β 3 integrin # ab75872 Abcam #A-10042 AlexaFluor568 ThermoFisher β 3 integrin vs Panx1 Panx1 #SC-49695 SantaCruz #A-11055 AlexaFluor488 ThermoFisher β 3 integrin vs P2X7R P2X7R # ab93354 Abcam #A-11055 AlexaFluor488 ThermoFisher β 3 integrin vs CaV3 Cav3 # SC-16263 SantaCruz #A-11055 AlexaFluor488 ThermoFisher β 3 integrin vs Cx43 Cx43 #C6219 Sigma #A-11034 AlexaFluor488 ThermoFisher Panx1 vs P2X7R, Panx1 #SC-49693 SantaCruz # {"type":"entrez-nucleotide","attrs":{"text":"A11055","term_id":"490909","term_text":"A11055"}} A11055 AlexaFluor488 ThermoFisher Panx1 vs CaV3 Panx1 #ab139715 Abcam #A-10042 AlexaFluor568 ThermoFisher Panx1 vs P2X7R P2X7R vs CaV3 P2X7R #ab109246 Abcam #A-10042 AlexaFluor568 ThermoFisher Panx1 vs CaV3 Cav3 #SC-16263 SantaCruz #A-11055 AlexaFluor488 ThermoFisher STORM FLAG F3165, mouse Sigma AlexaFluor488 Open in a separate window Antibodies Used in Co-Localization Studies SIM was performed using a Zeiss ELYRA S.1 system equipped with two laser channels; 1-2 μm z-axis image stacks were acquired in 110 nm optical steps (63× oil immersion objective, NA1.4).

Techniques: Fluorescence, Staining, Negative Control, Sampling

Journal: Journal of orthopaedic research : official publication of the Orthopaedic Research Society

Article Title: Potential Role for a Specialized β 3 Integrin-Based Structure On Osteocyte Processes in Bone Mechanosensation

doi: 10.1002/jor.23792

Figure Lengend Snippet: (A) Manders Coefficients for CaV3, P2X7R and Panx1 colocalizations with β3 integrin foci along osteocyte processes, plotted with respect to distance from the lacuna (p<0.05 for * vs β3 integrin with CaV3, P2X7R and Panx1 pair values) (B). Reverse Manders Coefficients for β3 integrin foci colocalizations with CaV3, P2X7R and Panx1along osteocyte processes, plotted with respect to distance from the lacunar margin. p<0.05 for * vs β3 integrin with CaV3, P2X7R and Panx1 pair values

Article Snippet: After staining, sections were mounted in Eukitt mounting media (EM Sciences) on precision thickness 1.5 glass coverslips (ThermoFisher). table ft1 table-wrap mode="anchored" t5 caption a7 EXPERIMENT TARGET 1°ANTIBODY VENDOR 2°ANTIBODY VENDOR Pilot β 3 integrin #ab20146 Abcam # A-32723 AlexaFluor488 ThermoFisher β 3 integrin vs Vinculin β 3 integrin # ab20146 Abcam #A-11019 AlexaFluor568 ThermoFisher β 3 integrin vs Vinculin Vinculin #V9131 Sigma #A-11034 AlexaFluor488 ThermoFisher β 3 integrin vs Panx1,P2X7R,CaV3 β 3 integrin # ab75872 Abcam #A-10042 AlexaFluor568 ThermoFisher β 3 integrin vs Panx1 Panx1 #SC-49695 SantaCruz #A-11055 AlexaFluor488 ThermoFisher β 3 integrin vs P2X7R P2X7R # ab93354 Abcam #A-11055 AlexaFluor488 ThermoFisher β 3 integrin vs CaV3 Cav3 # SC-16263 SantaCruz #A-11055 AlexaFluor488 ThermoFisher β 3 integrin vs Cx43 Cx43 #C6219 Sigma #A-11034 AlexaFluor488 ThermoFisher Panx1 vs P2X7R, Panx1 #SC-49693 SantaCruz # {"type":"entrez-nucleotide","attrs":{"text":"A11055","term_id":"490909","term_text":"A11055"}} A11055 AlexaFluor488 ThermoFisher Panx1 vs CaV3 Panx1 #ab139715 Abcam #A-10042 AlexaFluor568 ThermoFisher Panx1 vs P2X7R P2X7R vs CaV3 P2X7R #ab109246 Abcam #A-10042 AlexaFluor568 ThermoFisher Panx1 vs CaV3 Cav3 #SC-16263 SantaCruz #A-11055 AlexaFluor488 ThermoFisher STORM FLAG F3165, mouse Sigma AlexaFluor488 Open in a separate window Antibodies Used in Co-Localization Studies SIM was performed using a Zeiss ELYRA S.1 system equipped with two laser channels; 1-2 μm z-axis image stacks were acquired in 110 nm optical steps (63× oil immersion objective, NA1.4).

Techniques:

Journal: Purinergic Signalling

Article Title: Potential role of P2X7R in esophageal squamous cell carcinoma proliferation

doi: 10.1007/s11302-017-9559-2

Figure Lengend Snippet: Analysis of P2X receptor expression profile in human ESCC cell lines. Expression of purinergic receptors P2X1–7 in KYSE30, KYSE450, and OE21 cells lines by RT-PCR (a). GAPDH expression was used as reference gene. RT-qPCR analysis of P2X7R relative expression was performed, and the values were shown as ΔCq relative expression in relation to GAPDH in EPC2, KYSE30, KYSE450, and OE21 cell lines (b) and the significance was described as *p < 0.05 and *** p < 0.001 indicating difference in relation to KYSE450. Representative Western blotting assays showing positivity to P2X7R in EPC2, KYSE30, KYSE450, and KYSE520 cells (c). Relative P2X7R levels obtained by analysis of protein bands detected by Western blot were compared to GAPDH expression levels (d) and the significance was described as *p < 0.05 and *** p < 0.001 indicating difference in relation to EPC2. Each column represents the mean ± SEM and the experiments were performed three times in triplicate

Article Snippet: The nucleotides ATP, ADP, AMP, and adenosine as well the antagonist of P2X7R (A740003), unspecific antagonist of P2R (Pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt hydrate - PPADS and suramin), and apyrase were purchased from Sigma-Aldrich.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot

Journal: Purinergic Signalling

Article Title: Potential role of P2X7R in esophageal squamous cell carcinoma proliferation

doi: 10.1007/s11302-017-9559-2

Figure Lengend Snippet: Modulation of P2X7R reduces the effect cytotoxic generated by ATP in high concentrations. a Pharmacological modulation of P2X7R was performed by cell counting after treatment with A740003 10 μM, a specific antagonist of P2X7R, suramin 30 μM, and PPADS 10 μM as unspecific antagonist of P2X receptors and ATP 5 mM. The treatment was performed after sequential reduction of FBS concentration, and the cell counting was performed after 48 h posttreatment. The difference in relation to control was determined as **p < 0.01 and ***p < 0.001. The difference of ATP 5 mM was determined as #p < 0.05 and ###p < 0,001. b Representative Western blotting assays showing positivity to P2X7R in KYSE450 GFP−/− and KYSE450 siP2X7R. c Relative P2X7R levels obtained by analysis of protein bands detected by Western blot were compared to GAPDH expression levels and the significance was described as **p < 0.01 and indicated difference in relation to GFP−/−. d Effect of P2X7R silencing on cell viability. After 48 h of silencing, the cells were plated and MTT assays were performed following 24 h. The significance was described as ***p < 0.001 and indicated difference in relation to GFP−/−. e Effect of ATP (2.5 and 5 mM) treatment on siP2X7R cell viability. The experiments were performed three times in triplicate

Article Snippet: The nucleotides ATP, ADP, AMP, and adenosine as well the antagonist of P2X7R (A740003), unspecific antagonist of P2R (Pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt hydrate - PPADS and suramin), and apyrase were purchased from Sigma-Aldrich.

Techniques: Generated, Cell Counting, Concentration Assay, Western Blot, Expressing

Journal: Purinergic Signalling

Article Title: Potential role of P2X7R in esophageal squamous cell carcinoma proliferation

doi: 10.1007/s11302-017-9559-2

Figure Lengend Snippet: Immunostaining for P2X7 receptors in human esophageal samples. Representative images demonstrate the area of normal appearing esophageal mucosa with mild esophagitis (DAB, nuclear brown staining, ×200) (a), ESCC and glandular cells (b), and ESCC area (DAB, membrane brown staining, ×400) (c)

Article Snippet: The nucleotides ATP, ADP, AMP, and adenosine as well the antagonist of P2X7R (A740003), unspecific antagonist of P2R (Pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt hydrate - PPADS and suramin), and apyrase were purchased from Sigma-Aldrich.

Techniques: Immunostaining, Staining

Journal: Purinergic Signalling

Article Title: Up-regulation of P2X7 receptors mediating proliferation of Schwann cells after sciatic nerve injury

doi: 10.1007/s11302-015-9445-8

Figure Lengend Snippet: Expression of P2X7R ir in longitudinal sections of normal sciatic nerves. a–c show co-localization of P2X7R ir (red) and S100Aβ ir (green). Note an arrowhead showing a fibre-like structure and an arrow showing a trapezoid structure in a and b; c is the merged image of a and b. Note an arrow indicating a trapezoid structure double labelled by both P2X7R and S100β antibodies (yellow). d–f show co-localization of P2X7R ir (red) and Tuj-1 ir (green). Note an arrow showing a trapezoid structure in d and an arrow showing an axon in e. f is the merged image of d and f; note an arrow indicating a green axon passing through the middle of five trapezoid structures. g–i show co-localization of P2X7R ir (red) and p75NTR ir (green). Note an arrow showing a fibre-like structure in g and h. i is the merged image of g and h. An arrow indicates a double-labelled non-myelinating Schwann cell with P2X7R and p75NTR antibodies (yellow). j–l show co-localization of P2X7R ir (red) and MBP ir (green). Note an arrow showing a fibre-like structure with P2X7R ir, which was not labelled by MBP in l. m–o show co-localization of P2X7R ir (red) and CASPR ir (green). Note that no colocalization of P2X7R ir and CASPR ir was observed. Scale bars in a–c and g–i = 100 μm; scale bars in d–f = 50 μm

Article Snippet: The membranes were blocked with 10 % non-fat milk in Tris-buffered saline for 1 h and incubated overnight at 4 °C with P2X7R (1:1000, Alomone), p-ERK(1:1000, CST), ERK (1:1000, CST) and GAPDH (1:1000, Beyotime) antibodies diluted in 2 % BSA in PBS.

Techniques: Expressing

Journal: Purinergic Signalling

Article Title: Up-regulation of P2X7 receptors mediating proliferation of Schwann cells after sciatic nerve injury

doi: 10.1007/s11302-015-9445-8

Figure Lengend Snippet: Expression of P2X7R in teased sciatic nerve specimens. a–c show co-localization of P2X7R ir (red) and CASPR ir (green). Note an arrow showing a typical Ranvier node in a and b; c is the merged image of a and b. Note that the trapezoid structures with P2X7R ir were not labelled by CASPR ir. d–f show co-localization of P2X7R ir (red) and MBP ir (green). Note an arrow showing a Ranvier node with P2X7R ir in d and f. f is the merged image of d, e and an image of the same nerve fibre under phase-contrast microscopy; the arrow shows a typical Ranvier node with P2X7R ir. g–i show co-localization of P2X7R ir (red) and S100β ir (green). Note that the fibre-like structure with P2X7R ir was also labelled by S100β ir. j–l show co-localization of P2X7R ir and BMP ir. No colocalization of P2X7R ir and BMP ir was observed. All scale bars = 50 μm

Article Snippet: The membranes were blocked with 10 % non-fat milk in Tris-buffered saline for 1 h and incubated overnight at 4 °C with P2X7R (1:1000, Alomone), p-ERK(1:1000, CST), ERK (1:1000, CST) and GAPDH (1:1000, Beyotime) antibodies diluted in 2 % BSA in PBS.

Techniques: Expressing, Microscopy

Journal: Purinergic Signalling

Article Title: Up-regulation of P2X7 receptors mediating proliferation of Schwann cells after sciatic nerve injury

doi: 10.1007/s11302-015-9445-8

Figure Lengend Snippet: Expression of P2X7R (red) and S100β (green) in longitudinal sections after SNI. a is the merged image of a1 and a2, showing colocalization of P2X7R ir and S100β ir 2 days after SNI. Note that almost all the P2X7R ir cells are also labelled by S100β. b is the merged image of b1 and b2, showing colocalization of P2X7R ir and S100β ir 4 days after SNI. c is the merged image of c1 and c2, showing colocalization of P2X7R ir and S100β ir 7 days after SNI. d is the merged image of d1 and d2, showing colocalization of P2X7R ir and S100β ir 14 days after SNI. Note that almost all the P2X7R ir cells are also labelled by the S100β antibody in b, c and d. e is the merged image of e1 and e2, showing colocalization of P2X7R ir and S100β ir 30 days after SNI. Note that most of the P2X7R ir cells are also labelled by the S100β antibody, but some round cells with P2X7R ir were not labelled by the S100β antibody, as indicated by an arrow. f is the merged image of f1 and f2, showing colocalization of P2X7R ir and S100β ir 60 days after SNI. Scale bars in a–f = 100 μm; scale bars in all inserted small figures = 250 μm

Article Snippet: The membranes were blocked with 10 % non-fat milk in Tris-buffered saline for 1 h and incubated overnight at 4 °C with P2X7R (1:1000, Alomone), p-ERK(1:1000, CST), ERK (1:1000, CST) and GAPDH (1:1000, Beyotime) antibodies diluted in 2 % BSA in PBS.

Techniques: Expressing

Journal: Purinergic Signalling

Article Title: Up-regulation of P2X7 receptors mediating proliferation of Schwann cells after sciatic nerve injury

doi: 10.1007/s11302-015-9445-8

Figure Lengend Snippet: Expression of P2X7R detected by Western blotting in the sciatic nerves of normal and 2, 4, 7, 14, 30 and 60 d after SNI. a shows Western blots of P2X7 receptor protein (top panel) from the control group and 2, 4, 7, 14, 30 and 60 d after SNI. In the lower panel, blots were normalized to GAPDH to control for unequal protein loading between lanes. Data are representative of five rats/group. b The ratio of P2X7 receptor protein/GAPDH was analysed by one-way ANOVA followed by Dunnett’s post hoc test (*p < 0.05, **p < 0.01 vs. control group)

Article Snippet: The membranes were blocked with 10 % non-fat milk in Tris-buffered saline for 1 h and incubated overnight at 4 °C with P2X7R (1:1000, Alomone), p-ERK(1:1000, CST), ERK (1:1000, CST) and GAPDH (1:1000, Beyotime) antibodies diluted in 2 % BSA in PBS.

Techniques: Expressing, Western Blot

Journal: Purinergic Signalling

Article Title: Up-regulation of P2X7 receptors mediating proliferation of Schwann cells after sciatic nerve injury

doi: 10.1007/s11302-015-9445-8

Figure Lengend Snippet: Colocalization of P2X7R ir (red) and PCNA ir (green) in longitudinal sections of the sciatic nerve 2 days after SNI. a and b are P2X7R ir and PCNA ir cells, respectively. c is the merged image of a and b. d is the merged image of c and DAPI counterstaining (blue). An arrow indicates a typical cell with P2X7R ir, PCNA ir and DAPI counterstaining in a, b, c and d. All scale bars = 100 μm

Article Snippet: The membranes were blocked with 10 % non-fat milk in Tris-buffered saline for 1 h and incubated overnight at 4 °C with P2X7R (1:1000, Alomone), p-ERK(1:1000, CST), ERK (1:1000, CST) and GAPDH (1:1000, Beyotime) antibodies diluted in 2 % BSA in PBS.

Techniques:

Journal: Purinergic Signalling

Article Title: Up-regulation of P2X7 receptors mediating proliferation of Schwann cells after sciatic nerve injury

doi: 10.1007/s11302-015-9445-8

Figure Lengend Snippet: Expression of P2X7R in primary cultured Schwann cells (red). a shows the expression of P2X7R ir in normal Schwann cells. b shows the expression of P2X7R ir in Schwann cells stimulated with 100 μM/L ATP for 6 hours. Note that the expression of P2X7R was increased significantly. c shows the expression of P2X7R ir in Schwann cells stimulated with 100 μM/L ATP + 1 μM/L A740003 for 6 h. Note that the expression level of P2X7R is similar to that of the control group. d shows the expression of P2X7R ir in Schwann cells stimulated with 100 μM/L ATP + 2 μM/L AraC. Note that the expression level of P2X7R is similar to that of the control group. e is the result of Western blots: top panel is P2X7R protein from the control group, the ATP-stimulated group and the ATP + A740003 group. Blots were normalized to GAPDH (bottom panel) to control for unequal protein loading between lanes. Data are representative of five Schwann cell specimens/group. f is the ratio of P2X7R protein/GAPDH analysed by one-way ANOVA followed by Dunnett’s post hoc test (*p < 0.05, **p < 0.01). g is the result of Western blots: top panel is P2X7R protein from the control group, the ATP-stimulated group and the ATP + AraC group. Blots were normalized to GAPDH (bottom panel) to control for unequal protein loading between lanes. Data are representative of five Schwann cell specimens/group. h is the ratio of P2X7R protein/GAPDH analysed by one-way ANOVA followed by Dunnett’s post hoc test (*p < 0.05, **p < 0.01)

Article Snippet: The membranes were blocked with 10 % non-fat milk in Tris-buffered saline for 1 h and incubated overnight at 4 °C with P2X7R (1:1000, Alomone), p-ERK(1:1000, CST), ERK (1:1000, CST) and GAPDH (1:1000, Beyotime) antibodies diluted in 2 % BSA in PBS.

Techniques: Expressing, Cell Culture, Western Blot

Journal: Purinergic Signalling

Article Title: Up-regulation of P2X7 receptors mediating proliferation of Schwann cells after sciatic nerve injury

doi: 10.1007/s11302-015-9445-8

Figure Lengend Snippet: Assay of cell proliferation rates in primary cultured Schwann cells. a, d, g and j show BrdU-positive cells (green) in control, ATP, ATP + A740003 and ATP+ SCH772984 groups, respectively. b, e, h and k show counterstaining with DAPI of a, d, g and j. c, f, i and l are the merged images of a and b, d and e, g and h and j and k, respectively. m shows the statistical analysis of the Schwann cell proliferation rate. The results show that ATP increased significantly the Schwann cell proliferation rate, which was inhibited by the P2X7R antagonist A740003 and SCH772984. Scale bars = 40 μm (**p < 0.01)

Article Snippet: The membranes were blocked with 10 % non-fat milk in Tris-buffered saline for 1 h and incubated overnight at 4 °C with P2X7R (1:1000, Alomone), p-ERK(1:1000, CST), ERK (1:1000, CST) and GAPDH (1:1000, Beyotime) antibodies diluted in 2 % BSA in PBS.

Techniques: Cell Culture

Journal: The Journal of Biological Chemistry

Article Title: P2X7 receptor antagonism prevents IL-1β release from salivary epithelial cells and reduces inflammation in a mouse model of autoimmune exocrinopathy

doi: 10.1074/jbc.M117.790741

Figure Lengend Snippet: P2X7R activation induces the release of IL-1β in primary mouse SMG cell aggregates. A, dispersed SMG cell aggregates from wild-type mice were cultured in the presence or absence of A438079 (25 μm) for 30 min and then incubated with ATP (3 mm) or nigericin (10 μm) for 90 min (n = 4). B, dispersed SMG cell aggregates isolated from P2X7R−/− mice were incubated with ATP (3 mm) or nigericin (10 μm) for 90 min (n = 3). Cells were collected by centrifugation and IL-1β was quantified in the supernatant using the IL-1β Quantikine ELISA kit. Data represent means ± S.E., where **, p < 0.01 indicates a significant increase over basal levels, and ##, p < 0.01 indicates a significant decrease compared with ATP-treated cells.

Article Snippet: Male C57/BL6 (stock no. 000664) and P2X7R −/− (stock no. 005576) mice (6–8 weeks old) were purchased from The Jackson Laboratory (Bar Harbor, ME), whereas the P2X7R −/− mice (stock no. 005576) were originally donated by Pfizer Pharmaceuticals.

Techniques: Activation Assay, Cell Culture, Incubation, Isolation, Centrifugation, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: P2X7 receptor antagonism prevents IL-1β release from salivary epithelial cells and reduces inflammation in a mouse model of autoimmune exocrinopathy

doi: 10.1074/jbc.M117.790741

Figure Lengend Snippet: The NLRP3 inflammasome mediates P2X7R-induced IL-1β release. A, dispersed SMG cell aggregates from wild-type mice were cultured in the presence or absence of Bay 11–7082 (15 μm) for 1 h or MCC-950 (10 μm) for 30 min and then stimulated with ATP (3 mm) for 90 min (n = 3). Cells were collected by centrifugation and IL-1β was quantified in the supernatant using the IL-1β Quantikine ELISA kit. Data represent means ± S.E., where **, p < 0.01 and ***, p < 0.001 indicate significant decreases from ATP-treated only. B, upper panel, cell lysates from dispersed wild-type or P2X7R−/− SMG cell aggregates treated with or without ATP (3 mm) for 15 min were subjected to immunoprecipitation (IP) with anti-NLRP3 antibody, followed by immunoblotting (IB) with anti-NLRP3, anti-ASC, or anti–pro-caspase-1 antibodies. Images represent results from three independent experiments. Lower panel, quantification of the coimmunoprecipitated proteins in wild-type SMG cells (n = 3). Data represent means ± S.E., where *, p < 0.05 and **, p < 0.01 indicate significant increases in relative band intensities as compared with basal conditions.

Article Snippet: Male C57/BL6 (stock no. 000664) and P2X7R −/− (stock no. 005576) mice (6–8 weeks old) were purchased from The Jackson Laboratory (Bar Harbor, ME), whereas the P2X7R −/− mice (stock no. 005576) were originally donated by Pfizer Pharmaceuticals.

Techniques: Cell Culture, Centrifugation, Enzyme-linked Immunosorbent Assay, Immunoprecipitation, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: P2X7 receptor antagonism prevents IL-1β release from salivary epithelial cells and reduces inflammation in a mouse model of autoimmune exocrinopathy

doi: 10.1074/jbc.M117.790741

Figure Lengend Snippet: K+ efflux, ROS, and HSP90 are required for P2X7R-mediated IL-1β release. A, dispersed SMG cell aggregates from wild-type and P2X7R−/− mice were stimulated with ATP (3 mm) for 0, 10, or 30 min. Then, the intracellular [K+] was quantified using ICP-OES and expressed as a percentage of the intracellular [K+] at 0 min of ATP treatment (n = 3). Data represent means ± S.E., where *, p < 0.05 indicates a significant decrease from ATP-treated cells at 0 min. B, dispersed SMG cell aggregates from wild-type mice were stimulated with ATP (3 mm) for 90 min in the presence of the specified concentrations of NaCl, KCl, or choline chloride (n = 4). C–E, dispersed SMG cell aggregates from wild-type mice also were cultured in presence or absence of (C) the ROS inhibitor NAC (25 mm), (D) the mitochondrial ROS inhibitor Mito-Tempo (1 mm), or (E) the HSP90 inhibitor geldanamycin (GA) (5 μm) for 1 h and then stimulated with ATP (3 mm) for 90 min (n = 3). Cells were collected by centrifugation, and IL-1β was quantified in the supernatant using the IL-1β Quantikine ELISA kit. Data represent means ± S.E., where *, p < 0.05, **, p < 0.01, and ***, p < 0.001 indicate significant decreases from (B) ATP-treated cells in 145 mm Na+ or (C–E) ATP-treated only. F, upper panel, cell lysates from dispersed wild-type SMG cell aggregates were treated with or without GA (5 μm) for 1 h and subjected to immunoblotting with anti-NLRP3 or anti-β-tubulin antibodies. Images represent results from three independent experiments. Lower panel, quantification of NLRP3 expression in wild-type SMG cells (n = 3). Data represent means ± S.E., where *, p < 0.05 indicates a significant decrease in relative band intensity as compared with basal conditions.

Article Snippet: Male C57/BL6 (stock no. 000664) and P2X7R −/− (stock no. 005576) mice (6–8 weeks old) were purchased from The Jackson Laboratory (Bar Harbor, ME), whereas the P2X7R −/− mice (stock no. 005576) were originally donated by Pfizer Pharmaceuticals.

Techniques: Cell Culture, Centrifugation, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing

Journal: The Journal of Biological Chemistry

Article Title: P2X7 receptor antagonism prevents IL-1β release from salivary epithelial cells and reduces inflammation in a mouse model of autoimmune exocrinopathy

doi: 10.1074/jbc.M117.790741

Figure Lengend Snippet: P2X7R antagonism ameliorates salivary gland inflammation and reduces expression of IL-1β and immunoactive molecules in CD28−/−, IFNγ−/−, NOD.H-2h4 mice. A, H&E staining of SMG sections isolated from CD28−/−, IFNγ−/−, NOD.H-2h4 mice (5 months old) injected intraperitoneally with saline alone or the P2X7R antagonist A438079 (34.2 mg/kg/day) for 10 days. Images are representative of n = 10 mice per each group. B–E, cDNA was prepared from SMGs of NOD.H-2h4 and CD28−/−, IFNγ−/−, NOD.H-2h4 mice injected with saline alone or A438079, as above, then analyzed by RT-PCR using specific primers for (B) the pan-immune cell marker CD45 (n = 5 for NOD.H-2h4 mice; n = 9 for saline-injected; and n = 10 for A438079-injected CD28−/−, IFNγ−/−, NOD.H-2h4 mice); (C) IL-1β; (D) ICAM-1, VCAM, or E-Selectin; or (E) CD80 or CD86 (n = 4 for NOD.H-2h4 mice; n = 9 for saline-injected; and n = 10 for A438079-injected CD28−/−, IFNγ−/−, NOD.H-2h4 mice). Data represent means ± S.E., where *, p < 0.05 and **, p < 0.01 indicate significant differences from saline-injected CD28−/−, IFNγ−/−, NOD.H-2h4 mice.

Article Snippet: Male C57/BL6 (stock no. 000664) and P2X7R −/− (stock no. 005576) mice (6–8 weeks old) were purchased from The Jackson Laboratory (Bar Harbor, ME), whereas the P2X7R −/− mice (stock no. 005576) were originally donated by Pfizer Pharmaceuticals.

Techniques: Expressing, Staining, Isolation, Injection, Reverse Transcription Polymerase Chain Reaction, Marker

Journal: The Journal of Biological Chemistry

Article Title: P2X7 receptor antagonism prevents IL-1β release from salivary epithelial cells and reduces inflammation in a mouse model of autoimmune exocrinopathy

doi: 10.1074/jbc.M117.790741

Figure Lengend Snippet: P2X7R antagonism improves saliva flow rate in CD28−/−, IFNγ−/−, NOD. H-2h4 mice. Carbachol-induced saliva flow rate in CD28−/−, IFNγ−/−, NOD.H-2h4 mice (5 months old) injected with saline alone or the P2X7R antagonist A438079 (34.2 mg/kg/day) for 10 days (n = 9 for saline-injected and n = 10 for A438079-injected CD28−/−, IFNγ−/−, NOD.H-2h4 mice). Data represent means ± S.E., where **, p < 0.01 indicates a significant difference from saline-injected CD28−/−, IFNγ−/−, NOD.H-2h4 mice.

Article Snippet: Male C57/BL6 (stock no. 000664) and P2X7R −/− (stock no. 005576) mice (6–8 weeks old) were purchased from The Jackson Laboratory (Bar Harbor, ME), whereas the P2X7R −/− mice (stock no. 005576) were originally donated by Pfizer Pharmaceuticals.

Techniques: Injection

Journal: The Journal of Biological Chemistry

Article Title: P2X7 receptor antagonism prevents IL-1β release from salivary epithelial cells and reduces inflammation in a mouse model of autoimmune exocrinopathy

doi: 10.1074/jbc.M117.790741

Figure Lengend Snippet: Schematic diagram illustrating the proposed role of the P2X7R in salivary gland inflammation. In salivary epithelial cells, P2X7R activation by eATP induces the assembly and activation of the NLRP3 inflammasome and the subsequent release of mature IL-1β. This process involves transmembrane Na+ and/or K+ flux, production of ROS, and the presence of an active HSP90 protein. P2X7R antagonism reduces immune cell infiltration and salivary gland expression of IL-1β, ICAM-1, VCAM, E-Selectin, CD80, and CD86 and enhances carbachol-induced saliva secretion in the CD28−/−, IFNγ−/−, NOD.H-2h4 mouse model of salivary gland inflammation.

Article Snippet: Male C57/BL6 (stock no. 000664) and P2X7R −/− (stock no. 005576) mice (6–8 weeks old) were purchased from The Jackson Laboratory (Bar Harbor, ME), whereas the P2X7R −/− mice (stock no. 005576) were originally donated by Pfizer Pharmaceuticals.

Techniques: Activation Assay, Expressing