Journal: Journal of advanced research

Article Title: The purinergic receptor P2rx7 mediated ATP sensing is required to prevent bone aging by directing mitochondrial fitness of MSCs.

doi: 10.1016/j.jare.2025.06.026

Figure Lengend Snippet: Fig. 2. P2rx7 promoted the bone formation of MSCs. a The CFU-F in MSCs derived from the control and the P2rx7-/- MSCs. b Mineralized nodules formed by MSCs of the control and the P2rx7-/- mice were analyzed by alizarin red staining. c, d The expressions of osteogenesis marker genes in the control and the P2rx7-/- MSCs, as assessed by western blotting (c) and qPCR (d). e, f Transplants consisting of MSCs from control and P2rx7-/- mice mixed with HA/TCP were analyzed by H&E staining (e), and immunofluorescent staining of CD31 and ACAN (f), n = 5 per group. g Mineralized nodules formed by MSCs under osteogenic induction when exposed to 0, 40 nM or 100 nM BzATP were showed by alizarin red staining. h The expressions of osteogenic markers Runx2, ALP, and OCN in MSCs exposed to 0, 40 nM or 100 nM BzATP, as assessed by western blotting. i, j Transplants consisting of MSCs treated with 0, 40, 100 nM BzATP from control mice mixed with HA/TCP were analyzed by H&E staining (i), and immunofluorescent staining of CD31 and ACAN (j), n = 5 per group. Scale bar: e, f, i, j: 50 lm. e, i yellow, new bone; black, blood vessel; blue, new cartilage. Data was presented as mean ± SD. The data (a-d, g) are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs con by two-tailed Student’s t test (a, b, e, f) and one-way ANOVA (d, g, i, j). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The antibodies used in this study were as follows: Anti-mouse CD29 (#2473836), anti-mouse CD31 (#2373735), anti-mouse CD44 (#2255545) antibodies from eBioscience (San Diego, CA, USA); PE anti-mouse CD11b (#101207), APC anti-mouse CD90.2 (#105311) antibodies from BioLegend (San Diego, CA, USA); Anti-Col1a1 (ab255809; Abcam, Cambridge, MA, USA); Drp1 (#8570), Erk1/2 (#4370), p-Erk1/2 (#4695) from CST (Danvers, MA, USA); alkaline phosphatase (ALP, PA5-106391; Invitrogen, Carlsbad, CA, USA); Bmp-2 (sc-137087), CD90 (sc-53116), Cytoc (sc-13156), Ocn (sc-390877), P2rx7 (sc-514962) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Fis1 (10956–1-AP), p16 (10883–1-AP), p21 (10355–1-AP), P2rx7 (28207–1-AP), Mfn1 (13798–1-AP), Mfn2 (12186–1-AP), Opa1 (27733–1-AP), Runx2 (82636–2-RR) from ProteinTech (Cook, Illinois, USA); p-Drp1 (Ser616) (AF8470) from Affinity (Liyang, China); Hsp60 (AF0186) and Mouse IgG (A7028) from Beyotime (Shanghai, China); bActin (GB15001-100) from Servicebio (Wuhan, China).

Techniques: Derivative Assay, Control, Staining, Marker, Western Blot, Two Tailed Test

Journal: Journal of advanced research

Article Title: The purinergic receptor P2rx7 mediated ATP sensing is required to prevent bone aging by directing mitochondrial fitness of MSCs.

doi: 10.1016/j.jare.2025.06.026

Figure Lengend Snippet: Fig. 3. P2rx7 controls mitochondrial dynamics in MSCs. a OCR, calculated basal respiration and ATP-linked respiration of the control and the P2rx7-/- MSCs. b ATP contents of the control and the P2rx7-/- MSCs. c ECAR, and calculated glycolysis, glycolytic capability and glycolytic reverse of the control and the P2rx7-/- MSCs. d TEM images of mitochondria (arrows) in the control and the P2rx7-/- MSCs (n = 20 per group). e DWM of the control and the P2rx7-/- MSCs was measured by JC-1 staining (polymer: red; monomer: green). f The expressions of genes Nd1 and mt-Cytb in the control and the P2rx7-/- MSCs that related to the mitochondrial mass in cells assessed by qPCR. g DWM of MSCs treated with different concentrations of BzATP was analyzed by JC-1 staining. Scale bar: d: 500 nm; e, g: 100 lm. Data was presented as mean ± SD. The data (a, b, e-g) are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs con by two-tailed Student’s t test (a-f) and one-way ANOVA (g). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The antibodies used in this study were as follows: Anti-mouse CD29 (#2473836), anti-mouse CD31 (#2373735), anti-mouse CD44 (#2255545) antibodies from eBioscience (San Diego, CA, USA); PE anti-mouse CD11b (#101207), APC anti-mouse CD90.2 (#105311) antibodies from BioLegend (San Diego, CA, USA); Anti-Col1a1 (ab255809; Abcam, Cambridge, MA, USA); Drp1 (#8570), Erk1/2 (#4370), p-Erk1/2 (#4695) from CST (Danvers, MA, USA); alkaline phosphatase (ALP, PA5-106391; Invitrogen, Carlsbad, CA, USA); Bmp-2 (sc-137087), CD90 (sc-53116), Cytoc (sc-13156), Ocn (sc-390877), P2rx7 (sc-514962) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Fis1 (10956–1-AP), p16 (10883–1-AP), p21 (10355–1-AP), P2rx7 (28207–1-AP), Mfn1 (13798–1-AP), Mfn2 (12186–1-AP), Opa1 (27733–1-AP), Runx2 (82636–2-RR) from ProteinTech (Cook, Illinois, USA); p-Drp1 (Ser616) (AF8470) from Affinity (Liyang, China); Hsp60 (AF0186) and Mouse IgG (A7028) from Beyotime (Shanghai, China); bActin (GB15001-100) from Servicebio (Wuhan, China).

Techniques: Control, Staining, Polymer, Two Tailed Test

Journal: Journal of advanced research

Article Title: The purinergic receptor P2rx7 mediated ATP sensing is required to prevent bone aging by directing mitochondrial fitness of MSCs.

doi: 10.1016/j.jare.2025.06.026

Figure Lengend Snippet: Fig. 4. P2rx7 regulated mitochondrial fusion through Mfn1. a The mitochondria (top) labeled by Mitotracker Red and mitochondrial morphology (bottom) based on Mitochondrial Analyzer after grey processing, analyzed by Airyscan confocal images in the control and P2rx7-/- MSCs. b The expression of genes associated with mitochondrial dynamics in the control and the P2rx7-/- MSCs as assessed by western blotting. c Representative micrographs visualizing P2rx7 (arrows) on the mitochondria. d Reprehensive Airyscan confocal images of mitochondrial morphology (Mitotracker Red, top) and mitochondrial morphology (bottom) based on Mitochondrial Analyzer after grey processing in the MSCs treated with 0, 40 nM and 100 nM BzATP. e The expression of genes associated with mitochondrial dynamics in the MSCs treated with 0, 40 nM and 100 nM BzATP as assessed by western blotting. f Western blotting analysis of mitochondrial (left) and cytosolic (right) fractions of the MSCs treated with 40 nM and 100 nM BzATP. g IHC staining of Mfn1 and Opa1 proteins in the control and the P2rx7-/- MSCs that were subcutaneously implanted into immunocompromised mice with HA/TCP (n = 5 per group). Scale bar: a, d:10 lm (top), 5 lm(bottom); g: 100 lm; h: 500 lm. Data was presented as mean ± SD. The data (a, b, d-f) are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs con by two-tailed Student’s t test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The antibodies used in this study were as follows: Anti-mouse CD29 (#2473836), anti-mouse CD31 (#2373735), anti-mouse CD44 (#2255545) antibodies from eBioscience (San Diego, CA, USA); PE anti-mouse CD11b (#101207), APC anti-mouse CD90.2 (#105311) antibodies from BioLegend (San Diego, CA, USA); Anti-Col1a1 (ab255809; Abcam, Cambridge, MA, USA); Drp1 (#8570), Erk1/2 (#4370), p-Erk1/2 (#4695) from CST (Danvers, MA, USA); alkaline phosphatase (ALP, PA5-106391; Invitrogen, Carlsbad, CA, USA); Bmp-2 (sc-137087), CD90 (sc-53116), Cytoc (sc-13156), Ocn (sc-390877), P2rx7 (sc-514962) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Fis1 (10956–1-AP), p16 (10883–1-AP), p21 (10355–1-AP), P2rx7 (28207–1-AP), Mfn1 (13798–1-AP), Mfn2 (12186–1-AP), Opa1 (27733–1-AP), Runx2 (82636–2-RR) from ProteinTech (Cook, Illinois, USA); p-Drp1 (Ser616) (AF8470) from Affinity (Liyang, China); Hsp60 (AF0186) and Mouse IgG (A7028) from Beyotime (Shanghai, China); bActin (GB15001-100) from Servicebio (Wuhan, China).

Techniques: Labeling, Control, Expressing, Western Blot, Immunohistochemistry, Two Tailed Test

Journal: Journal of advanced research

Article Title: The purinergic receptor P2rx7 mediated ATP sensing is required to prevent bone aging by directing mitochondrial fitness of MSCs.

doi: 10.1016/j.jare.2025.06.026

Figure Lengend Snippet: Fig. 5. P2rx7 enhanced the sensitivity of ERK pathway to promote mitochondrial fusion. a Top 10 significantly enrichment of downregulated DEGs between control and P2rx7-/- MSCs analyzed by KEGG. b Expressions of Erk1/2 and p-Erk1/2 protein in the distal femurs were analyzed by IHC staining (n = 5 per group). c The phosphorylation level and the total expression level of Erk1/2 in the control and the P2rx7-/- MSCs before and after the activated by bFGF, as assessed by western blotting. d The phosphorylation level and the total expression level of Erk1/2 in MSCs treated with different concentrations of BzATP before and after the activated by 10 lM bFGF for 1 h, as assessed by western blotting. e Expressions of genes associated with mitochondrial dynamics in the Widetype (WT) and the P2rx7-/- MSCs treated with bFGF as assessed by western blotting. f Expressions of genes associated with mitochondrial dynamics in MSCs treated with siNC, siMfn1 and BzATP as assessed by western blotting. g DWM of the Widetype (WT) and the P2rx7-/-MSCs treated with BzATP and bFGF was analyzed by JC-1 staining. h ALP activity (top) and mineralized nodules (bottom) under osteogenic induction of human MSCs when exposed to 100 nM BzATP in the presence and absence of U0126. i The expression of osteogenesis marker genes in MSCs exposed with BzATP, in the presence and absence of 10 lM U0126. Scale bar: b: 100 lm; d: 50 lm; h: 500 lm. Data was presented as mean ± SD. The data (c-i) are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs con by two-tailed Student’s t test (b) and one-way ANOVA (g, h).

Article Snippet: The antibodies used in this study were as follows: Anti-mouse CD29 (#2473836), anti-mouse CD31 (#2373735), anti-mouse CD44 (#2255545) antibodies from eBioscience (San Diego, CA, USA); PE anti-mouse CD11b (#101207), APC anti-mouse CD90.2 (#105311) antibodies from BioLegend (San Diego, CA, USA); Anti-Col1a1 (ab255809; Abcam, Cambridge, MA, USA); Drp1 (#8570), Erk1/2 (#4370), p-Erk1/2 (#4695) from CST (Danvers, MA, USA); alkaline phosphatase (ALP, PA5-106391; Invitrogen, Carlsbad, CA, USA); Bmp-2 (sc-137087), CD90 (sc-53116), Cytoc (sc-13156), Ocn (sc-390877), P2rx7 (sc-514962) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Fis1 (10956–1-AP), p16 (10883–1-AP), p21 (10355–1-AP), P2rx7 (28207–1-AP), Mfn1 (13798–1-AP), Mfn2 (12186–1-AP), Opa1 (27733–1-AP), Runx2 (82636–2-RR) from ProteinTech (Cook, Illinois, USA); p-Drp1 (Ser616) (AF8470) from Affinity (Liyang, China); Hsp60 (AF0186) and Mouse IgG (A7028) from Beyotime (Shanghai, China); bActin (GB15001-100) from Servicebio (Wuhan, China).

Techniques: Control, Immunohistochemistry, Phospho-proteomics, Expressing, Western Blot, Staining, Activity Assay, Marker, Two Tailed Test

Journal: Journal of advanced research

Article Title: The purinergic receptor P2rx7 mediated ATP sensing is required to prevent bone aging by directing mitochondrial fitness of MSCs.

doi: 10.1016/j.jare.2025.06.026

Figure Lengend Snippet: Fig. 6. Bone metabolism and regeneration could be promoted by DCA treatment. a, b Increased mineralized nodules (a) and ALP activity (b) under osteogenic induction of the P2rx7-/- MSCs treated with PBS (Control) and 5 lM DCA. c, d Expressions of osteogenesis marker genes in the P2rx7-/- MSCs treated with Control and 5 lM DCA, as assessed by western blotting (c) and qPCR analysis (d). e, f Cross-sectional lCT images of the trabecular bone (e) and H&E staining of distal femurs (f) of 4-month-old P2rx7-/- mice treated with and without DCA, n = 5 per group. g, h Mouse mandibles of the Control and the BzATP group after 4 weeks of healing were presented as lCT images (g) and statistical analysis (g right, BV/TV in lCT images; h right, percent of area of bone tissue according to Masson staining), H&E staining (h, top) and Masson staining (h, bottom), n = 5 per group. Black arrow: new bone. i Expressions of Mfn1 and Opa1 proteins in the area of defect after 4-week healing were analyzed by IHC staining (n = 5 per group). Scale bar: f, h: 500 lm; i: 100 lm. Data was presented as mean ± SD. The data (a-d) are representative of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 vs con by two- tailed Student’s t test.

Article Snippet: The antibodies used in this study were as follows: Anti-mouse CD29 (#2473836), anti-mouse CD31 (#2373735), anti-mouse CD44 (#2255545) antibodies from eBioscience (San Diego, CA, USA); PE anti-mouse CD11b (#101207), APC anti-mouse CD90.2 (#105311) antibodies from BioLegend (San Diego, CA, USA); Anti-Col1a1 (ab255809; Abcam, Cambridge, MA, USA); Drp1 (#8570), Erk1/2 (#4370), p-Erk1/2 (#4695) from CST (Danvers, MA, USA); alkaline phosphatase (ALP, PA5-106391; Invitrogen, Carlsbad, CA, USA); Bmp-2 (sc-137087), CD90 (sc-53116), Cytoc (sc-13156), Ocn (sc-390877), P2rx7 (sc-514962) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); Fis1 (10956–1-AP), p16 (10883–1-AP), p21 (10355–1-AP), P2rx7 (28207–1-AP), Mfn1 (13798–1-AP), Mfn2 (12186–1-AP), Opa1 (27733–1-AP), Runx2 (82636–2-RR) from ProteinTech (Cook, Illinois, USA); p-Drp1 (Ser616) (AF8470) from Affinity (Liyang, China); Hsp60 (AF0186) and Mouse IgG (A7028) from Beyotime (Shanghai, China); bActin (GB15001-100) from Servicebio (Wuhan, China).

Techniques: Activity Assay, Control, Marker, Western Blot, Staining, Immunohistochemistry, Two Tailed Test

Journal: International Journal of Molecular Sciences

Article Title: Purinergic Receptors P2X7 and P2X4 as Markers of Disease Progression in the rd10 Mouse Model of Inherited Retinal Dystrophy

doi: 10.3390/ijms232314758

Figure Lengend Snippet: P2X7R expression in mouse retina. ( A ) Representative western blot and ( B ) protein quantification of P2X7R expression in C57BL/6J, rd10early, and rd10late mice. ( C ) Mean fluorescence values of P2X7R immunostaining in P2X7R-positive cells analyzed by flow cytometry in rd10early and rd10late mice compared with C57BL/6J mice. Student’s t -test * p < 0.05, ** p < 0.01. C57BL/6J1: postnatal day (P)20; C57BL/6J2: P40–P47; rd10early: P18 and rd10late: P44.

Article Snippet: Membranes were incubated at 4 °C overnight with the appropriate dilution of the following primary antibodies: rabbit anti-P2X7 receptor (extracellular) (1:200; Alomone Labs), rabbit anti-P2X4 receptor (extracellular) (1:200; Alomone Labs), and mouse anti-GAPDH (Sigma-Aldrich).

Techniques: Expressing, Western Blot, Fluorescence, Immunostaining, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Purinergic Receptors P2X7 and P2X4 as Markers of Disease Progression in the rd10 Mouse Model of Inherited Retinal Dystrophy

doi: 10.3390/ijms232314758

Figure Lengend Snippet: P2X7R expression in mouse retinas analyzed by immunohistochemistry. Cross-sectional cryosections of retinas from a representative C57BL/6J mouse ( A , D , G ) and rd10early ( B , E , H ) and rd10late ( C , F , I ) mice, stained with antibodies against P2X7R ( A – C , G – I ). Nuclei were stained with TO-PRO ( D – F ). In C57BL/6J mice, immunopositive fluorescence against P2X7R appears mainly located in the INL and GCL. RPE: retinal pigment epithelial cells; OS: outer segments; IS: inner segments; ONL: outer nuclear layer; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; GCL: ganglion cell layer. Scale bar: 50 µm.

Article Snippet: Membranes were incubated at 4 °C overnight with the appropriate dilution of the following primary antibodies: rabbit anti-P2X7 receptor (extracellular) (1:200; Alomone Labs), rabbit anti-P2X4 receptor (extracellular) (1:200; Alomone Labs), and mouse anti-GAPDH (Sigma-Aldrich).

Techniques: Expressing, Immunohistochemistry, Staining, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Purinergic Receptors P2X7 and P2X4 as Markers of Disease Progression in the rd10 Mouse Model of Inherited Retinal Dystrophy

doi: 10.3390/ijms232314758

Figure Lengend Snippet: P2X4R expression in mouse retina. ( A ) Representative western blot and ( B ) protein quantification of P2X4R expression in C57BL/6J, rd10early, and rd10late mice. ( C ) Mean fluorescence values of P2X4R immunostaining in P2X7R-positive cells analyzed by flow cytometry in rd10early and rd10late mice compared with the control expression in C57BL/6J mice. Student’s t -test, * p < 0.05. C57BL/6J1: postnatal day (P)20; C57BL/6J2: P40-P47; rd10early: P18 and rd10late: P44.

Article Snippet: Membranes were incubated at 4 °C overnight with the appropriate dilution of the following primary antibodies: rabbit anti-P2X7 receptor (extracellular) (1:200; Alomone Labs), rabbit anti-P2X4 receptor (extracellular) (1:200; Alomone Labs), and mouse anti-GAPDH (Sigma-Aldrich).

Techniques: Expressing, Western Blot, Fluorescence, Immunostaining, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Purinergic Receptors P2X7 and P2X4 as Markers of Disease Progression in the rd10 Mouse Model of Inherited Retinal Dystrophy

doi: 10.3390/ijms232314758

Figure Lengend Snippet: P2X7R and P2X4R expression in retinal myeloid cells (CD11b + ). The CD11b immunoreactive cell population was analyzed by flow cytometry. Bar graphs show the number of CD11b + cells expressing P2X7R ( A ) and the mean intensity of P2X7R fluorescence values ( B ). Also, the number of CD11b + cells expressing P2X4R is shown ( C ), and the mean intensity of P2X4R fluorescence values ( D ). C57BL/6J: postnatal day (P)47; rd10early: P18 and rd10late: P44. ** p < 0.01, *** p < 0.001.

Article Snippet: Membranes were incubated at 4 °C overnight with the appropriate dilution of the following primary antibodies: rabbit anti-P2X7 receptor (extracellular) (1:200; Alomone Labs), rabbit anti-P2X4 receptor (extracellular) (1:200; Alomone Labs), and mouse anti-GAPDH (Sigma-Aldrich).

Techniques: Expressing, Flow Cytometry, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Purinergic Receptors P2X7 and P2X4 as Markers of Disease Progression in the rd10 Mouse Model of Inherited Retinal Dystrophy

doi: 10.3390/ijms232314758

Figure Lengend Snippet: P2X7R and P2X4R expression in the CD11b + population, analyzed by flow cytometry. Whole retinal cells from C57BL/6J, rd10early, and rd10late mice were triple-stained with antibodies against CD11b, P2X7R, and P2X4R. After discarding the cell debris and selecting singlets, CD11b + cells were gated ( A ) and the expression of P2X7R and P2X4R was analyzed ( B – D ). ( B ) Double contour plots representing P2X7R and P2X4R expression, showing P2X7R- and P2X4R-positive populations (green line) and P2X7R- and P2X4R-highly immunoreactive populations (blue line). Each plot shows the sum of a minimum of 3 independent replicates. ( C , D ) Bar graphs showing the number of double-positive cells (P2X7R and P2X4R) and mean fluorescence values for P2X7R and P2X4R in the whole double-positive population ( C ) and the highly immunoreactive double-positive population ( D ). Student’s t -test, * p < 0.05, ** p < 0.01, *** p < 0.001, ns: non-statistically significant. C57BL/6J: postnatal day (P)47; rd10early: P18 and rd10late: P44.

Article Snippet: Membranes were incubated at 4 °C overnight with the appropriate dilution of the following primary antibodies: rabbit anti-P2X7 receptor (extracellular) (1:200; Alomone Labs), rabbit anti-P2X4 receptor (extracellular) (1:200; Alomone Labs), and mouse anti-GAPDH (Sigma-Aldrich).

Techniques: Expressing, Flow Cytometry, Staining, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Purinergic Receptors P2X7 and P2X4 as Markers of Disease Progression in the rd10 Mouse Model of Inherited Retinal Dystrophy

doi: 10.3390/ijms232314758

Figure Lengend Snippet: P2X7R and P2X4R expression analysis in CD11b+ cells, assessed by flow cytometry. Whole retinal cells from C57BL/6J, rd10early, and rd10late mice were triple-stained with antibodies against CD11b, P2X7R, and P2X4R. After discarding the cell debris and selecting singlets, CD11b-immunopositive cells were gated and two populations were selected according to medium or high immunoreactivity against the CD11b antibody, as shown in the contour plot (rd10 postnatal day [P]18) ( A ). The dot plot shows the sum of a minimum of 3 independent replicates. The immunoreactivity against P2X7R ( B ) and P2X4R ( C ) was compared in the two CD11b populations of each group of mice. Student’s t -test was performed between the two populations in each condition. Data are presented as mean values ± SD. Student’s t -test, ** p < 0.01. C57BL/6J: P47; rd10early: P18 and rd10late: P44.

Article Snippet: Membranes were incubated at 4 °C overnight with the appropriate dilution of the following primary antibodies: rabbit anti-P2X7 receptor (extracellular) (1:200; Alomone Labs), rabbit anti-P2X4 receptor (extracellular) (1:200; Alomone Labs), and mouse anti-GAPDH (Sigma-Aldrich).

Techniques: Expressing, Flow Cytometry, Staining

Journal: Biology Open

Article Title: P2X 7 regulates ependymo-radial glial cell proliferation in adult Danio rerio following spinal cord injury

doi: 10.1242/bio.060270

Figure Lengend Snippet: P2X 7 receptors were expressed by GFAP + ependymo-radial glial cells. Spinal cord cross sections are shown (dorsal is up; asterisk indicates central canal; scale bar: 50 μm). Representative images of GFAP and P2X 7 labelling in naïve fish (A-C′), at 7 dpi (D-F′), and at 14 dpi (G-I′). Colocalization (arrows) was seen in radial processes and cell bodies (C′,F′,I′). GFAP + cells that did not colocalize with P2X 7 were also detected (open arrowheads) (C′,F′,I′). n =3/group. Created with BioRender.com.

Article Snippet: This included a P2X 7 control antigen (Alomone Labs, BLP-PR004, Q64663).

Techniques:

Journal: Biology Open

Article Title: P2X 7 regulates ependymo-radial glial cell proliferation in adult Danio rerio following spinal cord injury

doi: 10.1242/bio.060270

Figure Lengend Snippet: P2X 7 receptors were expressed by PCNA + proliferating cells. Spinal cord cross sections are shown (dorsal is up; asterisk indicates central canal; scale bar: 50 μm). Representative images of PCNA + and P2X 7 labelling in naïve fish (A-C′), at 7 dpi rostral (D-F′), and at 7 dpi caudal (G-I′). Colocalization (arrows) was seen in some cells (C′,F′,I′). PCNA + cells that did not colocalize with P2X 7 were also detected (open arrowheads) (C′,F′,I′). n =3/group. Created with BioRender.com.

Article Snippet: This included a P2X 7 control antigen (Alomone Labs, BLP-PR004, Q64663).

Techniques:

Journal: Biology Open

Article Title: P2X 7 regulates ependymo-radial glial cell proliferation in adult Danio rerio following spinal cord injury

doi: 10.1242/bio.060270

Figure Lengend Snippet: P2X 7 receptors were expressed by HuC/D + neurons. Spinal cord cross sections are shown (dorsal is up; asterisk indicates central canal; scale bar: 50 μm). Representative images of HuC/D + and P2X 7 labelling in naïve fish (A-C′), at 14 dpi rostral (D-F′), and at 14 dpi caudal (G-I′). Colocalization (arrows) was seen in larger mature neurons and smaller immature neurons (C′,F′,I′). n =3/group. Created with BioRender.com.

Article Snippet: This included a P2X 7 control antigen (Alomone Labs, BLP-PR004, Q64663).

Techniques:

Journal: Biology Open

Article Title: P2X 7 regulates ependymo-radial glial cell proliferation in adult Danio rerio following spinal cord injury

doi: 10.1242/bio.060270

Figure Lengend Snippet: Temporal changes in P2X 7 protein expression following SCI in adult zebrafish. Schematic representations of naïve and injured spinal cord areas of analysis (A-B), as well as tissue collection timeline (C). Representative western blot and corresponding total protein in naïve zebrafish (Zf) spinal cord tissue, mouse (Ms) hippocampal tissue as a positive control (+), and pre-absorption of naïve zebrafish spinal cord tissue with a blocking peptide as a negative control (-) (D). Representative western blot and corresponding total protein at 1 dpi (E), 7 dpi (G), and 14 dpi (I). Quantitative analysis of the 50 kDa isoform showed significant downregulation within the injury (In, n =10, one-way ANOVA with Dunnett's post hoc test, P =0.0079) and caudal to the lesion (Ca, n =9, P =0.0046) at 7 dpi when normalized to naïve tissue ( n =9) (H). Protein expression of the 50 kDa isoform showed basal levels of expression at 1 dpi (F) and 14 dpi (J). Data presented as means±s.d. **, significant differences, P <0.01. Created with BioRender.com.

Article Snippet: This included a P2X 7 control antigen (Alomone Labs, BLP-PR004, Q64663).

Techniques: Expressing, Western Blot, Positive Control, Blocking Assay, Negative Control

Journal: iScience

Article Title: Hepatitis B virus-mediated sodium influx contributes to hepatic inflammation via synergism with intrahepatic danger signals

doi: 10.1016/j.isci.2023.108723

Figure Lengend Snippet:

Article Snippet: anti-P2X7 , Cell signaling Techonology , 13809.

Techniques: Activity Assay, ATP Assay, Enzyme-linked Immunosorbent Assay, Sequencing, Plasmid Preparation, RNA Sequencing Assay, Biomarker Assay, Software

Journal: International Journal of Molecular Sciences

Article Title: Overexpression of P2X3 and P2X7 Receptors and TRPV1 Channels in Adrenomedullary Chromaffin Cells in a Rat Model of Neuropathic Pain

doi: 10.3390/ijms20010155

Figure Lengend Snippet: CCI animals show increased P2X3 and P2X7 receptor immunoreactivity in DRG and mechanical allodynia involving P2X3 receptors. ( A ) Fluorescence confocal images of DRG from control (upper panels) and CCI animals (lower panels) stained for P2X3 receptors and glial fibrillary acidic protein (GFAP). ( B ) Fluorescence confocal images of DRG from control (upper panels) and CCI animals (lower panels) stained for P2X7 receptors and GFAP. Cell nuclei in A and B were visualized following incubation with 4′6-diamidino-2-phenylindole (DAPI). Images are representative of those obtained in DRG from 3 Control and 3 CCI animals. ( C ) Effect of intraplantar (i.pl.) administration of diadenosine polyphosphates on paw withdrawal threshold to mechanical stimulation. Left ; Effect of i.pl. administration of Ap 5 A (2 µg; n = 6 experiments, 3 rats), Ip 5 I (2 µg; n = 8 experiments, 4 rats), and co-administration of Ap 5 A and Ip 5 I ( n = 6 experiments, 3 rats) in the hindpaw of control animals. Right ; Effect of i.pl. administration of Ap 4 A (2 µg; n = 3 experiments, 3 rats), Ip 5 I (2 µg; n = 8 experiments, 4 rats), and co-administration of Ap 4 A and Ip 5 I ( n = 4 experiments, 4 rats) in the ipsilateral hindpaw of CCI animals. Statistical significance was assessed by a Student t test for unpaired data with respect to vehicle (Veh.; ***, p < 0.001), and for paired data with respect to AP 5 A ( ## , p < 0.01).

Article Snippet: Sections were permeabilized for 1 h at room temperature with 0.2% Triton-X100/2% bovine serum albumin (BSA) in phosphate buffer, rinsed with 2% BSA in phosphate buffer and incubated overnight at 4 °C with the following combinations of primary antibodies: (a) rabbit anti-P2X3 (1:100; Alomone, Jerusalem, Israel) and guinea-pig anti-TH (1:500; Synaptic Systems, Goettingen, Germany); (b) rabbit anti-P2X7 (1:100; Alomone) and anti-TH (1:500); (c) goat antibodies against the VAChT (1:50; Santa Cruz Biotechnology, Madrid, Spain) were used either alone or in combination with anti-P2X7 or -P2X3 antibodies; and (d) rabbit anti-TRPV1 (1:100; Abcam, Cambridge, UK) and anti-TH (1:500).

Techniques: Fluorescence, Staining, Incubation

Journal: International Journal of Molecular Sciences

Article Title: Overexpression of P2X3 and P2X7 Receptors and TRPV1 Channels in Adrenomedullary Chromaffin Cells in a Rat Model of Neuropathic Pain

doi: 10.3390/ijms20010155

Figure Lengend Snippet: Functional plasticity of P2X3 receptors in chromaffin cells from CCI animals. ( A ) Voltage-clamp recordings of currents evoked by α,β-meATP in chromaffin cells of the adrenal gland from Control animals. The horizontal bars show the application of α,β-meATP (10 μM, 3 s), which was applied twice with a 2 min interval (+2’ wash). Representative recordings of rapidly desensitizing (left panel) and non-desensitizing (right panel) currents. ( B ) Sensitivity to Ip 5 I or to A438079 (a selective P2X7 receptor blocker) of currents evoked by α,β-meATP in chromaffin cells from Control animals. Horizontal bars show application of α,β-meATP (10 μM, 3 s), which was applied twice with a 10 min interval, in the absence or the presence of Ip 5 I (+Ip 5 I; 10 μM, 2 min; left panel) or A438079 (+A438079; 10 μM, 2 min; right panel). ( C ) Similar to B but referred to CCI animals. Scale bars apply to panels ( A – C ). ( D ). Amplitudes of 10 µM α,β-meATP-evoked currents in chromaffin cells from Control and CCI animals in the absence and the presence of Ip 5 I (+Ip 5 I; 10 μM) or A438079 ((+A438079; 10 μM). Data from the number of cells are shown between parentheses on top of bars. Statistical significances were assessed by a Student t test for paired data when evaluating the effect of receptor antagonists (*, p < 0.05; **, p < 0.01), and for unpaired data when evaluating the effect of CCI ( ## , p < 0.01).

Article Snippet: Sections were permeabilized for 1 h at room temperature with 0.2% Triton-X100/2% bovine serum albumin (BSA) in phosphate buffer, rinsed with 2% BSA in phosphate buffer and incubated overnight at 4 °C with the following combinations of primary antibodies: (a) rabbit anti-P2X3 (1:100; Alomone, Jerusalem, Israel) and guinea-pig anti-TH (1:500; Synaptic Systems, Goettingen, Germany); (b) rabbit anti-P2X7 (1:100; Alomone) and anti-TH (1:500); (c) goat antibodies against the VAChT (1:50; Santa Cruz Biotechnology, Madrid, Spain) were used either alone or in combination with anti-P2X7 or -P2X3 antibodies; and (d) rabbit anti-TRPV1 (1:100; Abcam, Cambridge, UK) and anti-TH (1:500).

Techniques: Functional Assay

Journal: International Journal of Molecular Sciences

Article Title: Overexpression of P2X3 and P2X7 Receptors and TRPV1 Channels in Adrenomedullary Chromaffin Cells in a Rat Model of Neuropathic Pain

doi: 10.3390/ijms20010155

Figure Lengend Snippet: Functional plasticity of P2X7 receptors in chromaffin cells from CCI animals. ( A ) Voltage-clamp recordings of currents evoked by BzATP (a P2X7 receptor agonist) in chromaffin cells of the adrenal gland from Control animals. The horizontal bars show the application of BzATP (100 μM, 3 s), which was applied twice with a 10 min interval in the absence or the presence of A438079 (+A438079; 10 μM, 2 min; left panel) or of Ip 5 I (+Ip 5 I; 10 μM, 2 min; right panel). ( B ) Similar to panel ( A ), but referred to CCI animals. Scale bars apply to panels ( A , B ). ( C ) Amplitudes of 100 µM BzATP-evoked currents in chromaffin cells from Control and CCI animals in the absence and the presence of Ip 5 I ((+Ip 5 I; 10 μM) or A438079 (+A438079; 10 μM). Data from the number of cells are shown between parentheses on top of bars. Statistical significances were assessed by a Student t test for paired data when evaluating the effect of receptor antagonists (*, p < 0.05; **, p < 0.01), and unpaired data when evaluating the effect of CCI ( ## , p < 0.01).

Article Snippet: Sections were permeabilized for 1 h at room temperature with 0.2% Triton-X100/2% bovine serum albumin (BSA) in phosphate buffer, rinsed with 2% BSA in phosphate buffer and incubated overnight at 4 °C with the following combinations of primary antibodies: (a) rabbit anti-P2X3 (1:100; Alomone, Jerusalem, Israel) and guinea-pig anti-TH (1:500; Synaptic Systems, Goettingen, Germany); (b) rabbit anti-P2X7 (1:100; Alomone) and anti-TH (1:500); (c) goat antibodies against the VAChT (1:50; Santa Cruz Biotechnology, Madrid, Spain) were used either alone or in combination with anti-P2X7 or -P2X3 antibodies; and (d) rabbit anti-TRPV1 (1:100; Abcam, Cambridge, UK) and anti-TH (1:500).

Techniques: Functional Assay

Journal: International Journal of Molecular Sciences

Article Title: Overexpression of P2X3 and P2X7 Receptors and TRPV1 Channels in Adrenomedullary Chromaffin Cells in a Rat Model of Neuropathic Pain

doi: 10.3390/ijms20010155

Figure Lengend Snippet: Increased P2X7 receptor immunoreactivity in chromaffin cells from CCI animals. ( A ) Fluorescence confocal images of adrenal gland slices from Control (upper panels) and CCI animals (lower panels) stained for P2X7 receptor and tyrosine hydroxylase (TH). ( B ) Fluorescence confocal images of adrenal gland slices from Control (upper panels) and CCI animals (lower panels) stained for P2X7 receptor and the vesicular acetylcholine transporter (VAChT). Cell nuclei in ( A , B ) were visualized following incubation with DAPI. Images are representative of those obtained from 3 Control and 3 CCI animals.

Article Snippet: Sections were permeabilized for 1 h at room temperature with 0.2% Triton-X100/2% bovine serum albumin (BSA) in phosphate buffer, rinsed with 2% BSA in phosphate buffer and incubated overnight at 4 °C with the following combinations of primary antibodies: (a) rabbit anti-P2X3 (1:100; Alomone, Jerusalem, Israel) and guinea-pig anti-TH (1:500; Synaptic Systems, Goettingen, Germany); (b) rabbit anti-P2X7 (1:100; Alomone) and anti-TH (1:500); (c) goat antibodies against the VAChT (1:50; Santa Cruz Biotechnology, Madrid, Spain) were used either alone or in combination with anti-P2X7 or -P2X3 antibodies; and (d) rabbit anti-TRPV1 (1:100; Abcam, Cambridge, UK) and anti-TH (1:500).

Techniques: Fluorescence, Staining, Incubation

Journal: Purinergic Signalling

Article Title: P2X7 receptor: the regulator of glioma tumor development and survival

doi: 10.1007/s11302-021-09834-2

Figure Lengend Snippet: Characterization of P2X7 receptor in glioma C6 cells in vitro. a Western blot analysis of P2X7 protein expression in C6 cells after P2X7 silencing for 72 h ( n = 3). b Calcium signal level in glioma C6 cells after P2X7 downregulation and BzATP stimulation (300 µM) ( n = 4). Gray lines represent the signals from the biological replicates; black lines represent the signal averaged among the individual replicates. Insert shows effect of P2X7 downregulation on the signal strength, one-way t -test: * P ≤ 0.05. c Measurement of bioluminescence originated from released ATP after P2X7 activation/inhibition in C6 cells ( n = 3). The statistical significance of the differences was determined with one-way ANOVA with Bonferroni post hoc test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. control group

Article Snippet: Three target-specific RNAi (small interfering RNA) complementary to rat P2X7 transcript (sc-108056) and control non-silencing RNAi were obtained from Santa Cruz Biotechnology, Inc. (USA).

Techniques: In Vitro, Western Blot, Expressing, Activation Assay, Inhibition, Control

Journal: Purinergic Signalling

Article Title: P2X7 receptor: the regulator of glioma tumor development and survival

doi: 10.1007/s11302-021-09834-2

Figure Lengend Snippet: P2X7 activation increased C6 cell proliferation, viability, and cell adhesion in vitro. Quantitative data are presented as signal relative to control. a MTS test of long-term C6 cell viability after 100 µM BzATP stimulation of control cells and cells with P2X7 downregulation using RNAi ( n = 3). The significance of the differences was determined with paired t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control. b Representative Western blot analysis of pro-survival and pro-proliferative proteins: p-p38 (Thr180/Tyr182), pAkt (Ser473), tAkt, CD133, HSPA1, and HSPA5 in C6 cell lysates after 24-h BzATP stimulation ( n = 3). c Upper panel: MTS test of C6 cell viability after 200 µM BCNU (carmustine) treatment for 24 h. Co-treatment with 100 µM BzATP and 200 µM BCNU increased viability of C6 cells ( n = 3). The significance of the differences was determined with one-way ANOVA with Bonferroni post hoc test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. all groups. Bottom panel: a crystal violet assay demonstrating a synergistic effect of BBG with BCNU/TMZ co-treatment on C6 cell growth for 24 h ( n = 6). The significance of the differences was determined with repeated measures one-way ANOVA with Duncan’s multiple range post hoc test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. all groups. d P2X7 activation affects C6 cell adhesion to extracellular matrix components: collagen I, collagen IV, fibronectin. C6 cells showed higher adhesion potential after 24-h BzATP stimulation. 24-h co-treatment/treatment with 100 nM BBG significantly decreased C6 cell adhesion to ECM components ( n = 6). The significance of the differences was determined with one-way ANOVA with Bonferroni post hoc test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. control group. e P2X7 RNAi downregulation declined C6 cell adhesion to ECM components upon BzATP stimulation ( n = 4). The significance of the differences was determined with paired t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control

Article Snippet: Three target-specific RNAi (small interfering RNA) complementary to rat P2X7 transcript (sc-108056) and control non-silencing RNAi were obtained from Santa Cruz Biotechnology, Inc. (USA).

Techniques: Activation Assay, In Vitro, Control, Western Blot, Crystal Violet Assay

Journal: Purinergic Signalling

Article Title: P2X7 receptor: the regulator of glioma tumor development and survival

doi: 10.1007/s11302-021-09834-2

Figure Lengend Snippet: Activation of P2X7 receptor led to elevated ROS production and mitochondrial membrane depolarization in C6 cells. Quantitative data are presented as signal relative to control. a DCF-DA fluorescence measurement in C6 glioma cells. Cells stimulated with 100 µM BzATP for 1 h were characterized by increased ROS production compared to control in C6 cells ( n = 3). The significance of the differences was determined with one-way ANOVA with Bonferroni post hoc test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. control group. b Potential-dependent staining of mitochondria in C6 cells using JC-1. 1-h BzATP (100 µM) stimulation led to considerable mitochondrial membrane depolarization ( n = 3). The significance of the differences was determined with one-way ANOVA with Bonferroni post hoc test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. control group. c Representative images of MitoTracker Red CMXRos accumulation in C6 cells stimulated with 100 µM BzATP for 1 h. d DCF-DA fluorescence measurement in C6 glioma cells with RNA interference of P2X7 expression ( n = 4). Downregulation of P2X7 led to decreased ROS production in C6 cells after 1-h BzATP (100 µM) stimulation. The significance of the differences was determined using paired t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control. e Potential-dependent staining of mitochondria using JC-1 dye in C6 cells with RNA interference of P2X7 expression after 1 h of 100 μM BzATP pre-treatment ( n = 3). The amount of depolarized mitochondria was higher in control cells when compared to that in cells with P2X7 downregulation. The significance of the differences was determined using paired t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control

Article Snippet: Three target-specific RNAi (small interfering RNA) complementary to rat P2X7 transcript (sc-108056) and control non-silencing RNAi were obtained from Santa Cruz Biotechnology, Inc. (USA).

Techniques: Activation Assay, Membrane, Control, Fluorescence, Staining, Expressing

Journal: Purinergic Signalling

Article Title: P2X7 receptor: the regulator of glioma tumor development and survival

doi: 10.1007/s11302-021-09834-2

Figure Lengend Snippet: Effect of P2X7 inhibition using BBG on glioma C6 tumor development in vivo. a Brilliant Blue G administration led to reduction of glioma tumor mass. Representative images of control and BBG-treated C6 glioma tumors ( n = 9 for control group and n = 10 for BBG-treated group). The significance of the differences was determined using Student’s t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control. b Representative Western blot showing the significant decrease of P2X7 expression in tumor homogenates after BBG treatment. Representative image of P2X7 immunodetection in C6 glioma tumors. c P2X7 inhibition resulted in diminished activation of matrix metalloproteinase-2 in glioma tumors. The significance of the differences was determined with Student’s t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control. d Western blot analysis of known proteins involved in cell adhesion and EMT signaling in C6 tumor homogenates ( n = 4). e Western blot analysis of known proteins involved in tumor aggressiveness in C6 tumor homogenates ( n = 4)

Article Snippet: Three target-specific RNAi (small interfering RNA) complementary to rat P2X7 transcript (sc-108056) and control non-silencing RNAi were obtained from Santa Cruz Biotechnology, Inc. (USA).

Techniques: Inhibition, In Vivo, Control, Western Blot, Expressing, Immunodetection, Activation Assay

Journal: Purinergic Signalling

Article Title: P2X7 receptor: the regulator of glioma tumor development and survival

doi: 10.1007/s11302-021-09834-2

Figure Lengend Snippet: P2X7 inhibition reduced the ATP release with simultaneous decrease of some pro-inflammatory cytokine levels in mouse serum and changed the redox metabolism in C6 tumors. a An ATP release is significantly lower in tumors treated with BBG compared to the control tumors ( n = 4). The significance of the differences was determined using Student’s t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control. b Decline NOS-2 expression in BBG-treated tumors ( n = 4). c Decreased expression of FOXP3 (T reg lymphocytes) and CD68 (macrophages) markers in BBG-treated tumors evaluated using flow cytometry ( n = 5). The significance of the differences was determined using Student’s t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control. d The level of the pro-inflammatory and anti-inflammatory cytokines, in mouse serum and glioma tumors using cytokine array for multiplex protein detection ( n = 3). e P2X7 inhibition caused a decrease of GSSG/GSH ratio with a slight decline of SOD activity in BBG-treated tumors ( n = 4). The significance of the differences was determined using Student’s t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control

Article Snippet: Three target-specific RNAi (small interfering RNA) complementary to rat P2X7 transcript (sc-108056) and control non-silencing RNAi were obtained from Santa Cruz Biotechnology, Inc. (USA).

Techniques: Inhibition, Control, Expressing, Flow Cytometry, Multiplex Assay, Activity Assay

Journal: Purinergic Signalling

Article Title: P2X7 receptor: the regulator of glioma tumor development and survival

doi: 10.1007/s11302-021-09834-2

Figure Lengend Snippet: P2X7 receptor effects on human glioma development. a Western blot analysis of p-p38 MAPK, CD-133 and HSPA1 expression in U-138 human glioma cells ( n = 3). b In silico analysis of P2X7 expression level and survival of glioma patients using information from the TCGA database. c In silico analysis of P2X7 expression level in different WHO astrocytoma grades compared to normal tissue using NCBI dataset. d A transwell migration assay of LN-229 and U-251 glioma cells treated with 2 μM KN-62 for 24 h ( n = 3). e A crystal violet assay demonstrating a synergistic effect of 2 μM KN-62 with BCNU/TMZ co-treatment on LN-229, U-251, and U-138 glioma cell growth for 24 h ( n = 4). The significance of the differences was determined with repeated-measures one-way ANOVA with Duncan multiple range post hoc test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. all groups

Article Snippet: Three target-specific RNAi (small interfering RNA) complementary to rat P2X7 transcript (sc-108056) and control non-silencing RNAi were obtained from Santa Cruz Biotechnology, Inc. (USA).

Techniques: Western Blot, Expressing, In Silico, Transwell Migration Assay, Crystal Violet Assay

Journal: PLoS ONE

Article Title: P2X 4 Assembles with P2X 7 and Pannexin-1 in Gingival Epithelial Cells and Modulates ATP-induced Reactive Oxygen Species Production and Inflammasome Activation

doi: 10.1371/journal.pone.0070210

Figure Lengend Snippet: (A) Immortalized GEC that had been depleted of P2X 4 or P2X 7 using lentiviral particles were treated with 100 µM or 3 mM ATP for 3 hours, and supernatants were collected for caspase-1 activity measurement. Caspase-1 activity was measured by ELISA as described in . (B) Total proteins isolated from GEC were subjected to immunoprecipitation (IP) by a polyclonal anti-P2X 4 antibody or Dynabeads as a control. Precipitates or total protein extract (as input) were resolved on SDS-PAGE and analyzed on immunoblots with anti-P2X 7 (top), anti-pannexin-1 (middle), or anti-P2X 4 (bottom) antibodies.

Article Snippet: The siRNA sequences were: 5′-GCUUUCAACGGGUCUGUCATT-3′ and 5′-UGACAGACCCGUUGAAAGCTA-3′ for P2X 4 (Ambion, LifeCell Technologies, S9957, Cat. #: 4392420); and 5′-ACAAUGUUGAGAAACGGACUCUGAT-3′ for P2X 7 (27 mer siRNA duplexes OriGene Technologies, Cat. #: SR303325).

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Isolation, Immunoprecipitation, SDS Page, Western Blot

Journal: PLoS ONE

Article Title: P2X 4 Assembles with P2X 7 and Pannexin-1 in Gingival Epithelial Cells and Modulates ATP-induced Reactive Oxygen Species Production and Inflammasome Activation

doi: 10.1371/journal.pone.0070210

Figure Lengend Snippet: Primary GEC (C and D) and immortalized GEC (A and B) were infected with or without P. gingivalis ( P.g. ) at an M.O.I. of 100 for 6 hours, followed by treatment with different pharmaceutical agents. Infected cells were treated with 100 µM ATP, 3 mM ATP, 3 mM ADP, 3 mM AMP, or 3 mM UTP individually for 1 hour (A and C). Alternatively, infected cells were pre-treated with 50 µM 5-BDBD for 15 minutes, 100 µM PPADS for 15 minutes, 100 µM oxATP for 30 minutes, or 1 mM probenecid for 10 minutes, followed by treatment with 3 mM ATP for 1 hour (B and D). The supernatants were collected and subjected to ELISA to measure IL-1β secretion. (E) Primary GEC were transfected with siRNA sequences against P2X 4 or P2X 7 for one day, and mRNA levels were detected by qPCR. (F) Primary GEC depleted of P2X 4 or P2X 7 were infected with P. gingivalis ( P.g. ) and treated with probenecid and 3 mM ATP as shown in (B). IL-1β secretion in the supernatants was analyzed by ELISA. The values showed averages and SD from duplicate samples, which were obtained from three separate experiments.

Article Snippet: The siRNA sequences were: 5′-GCUUUCAACGGGUCUGUCATT-3′ and 5′-UGACAGACCCGUUGAAAGCTA-3′ for P2X 4 (Ambion, LifeCell Technologies, S9957, Cat. #: 4392420); and 5′-ACAAUGUUGAGAAACGGACUCUGAT-3′ for P2X 7 (27 mer siRNA duplexes OriGene Technologies, Cat. #: SR303325).

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Transfection

Journal: PLoS ONE

Article Title: P2X 4 Assembles with P2X 7 and Pannexin-1 in Gingival Epithelial Cells and Modulates ATP-induced Reactive Oxygen Species Production and Inflammasome Activation

doi: 10.1371/journal.pone.0070210

Figure Lengend Snippet: Model showing the role of P2X 4 and P2X 7 in ROS production and inflammasome activation in GEC stimulated with extracellular ATP.

Article Snippet: The siRNA sequences were: 5′-GCUUUCAACGGGUCUGUCATT-3′ and 5′-UGACAGACCCGUUGAAAGCTA-3′ for P2X 4 (Ambion, LifeCell Technologies, S9957, Cat. #: 4392420); and 5′-ACAAUGUUGAGAAACGGACUCUGAT-3′ for P2X 7 (27 mer siRNA duplexes OriGene Technologies, Cat. #: SR303325).

Techniques: Activation Assay