Journal: bioRxiv

Article Title: NINJ1 is activated by calcium-driven plasma membrane lipid scrambling during lytic cell death

doi: 10.1101/2024.10.23.619800

Figure Lengend Snippet: A, Stimulation of peritoneal macrophage with BzATP induces lytic cell death as assayed by LDH release into the supernatant. Pretreatment with A238079 prevents BzATP-induced cytotoxicity. * p < 0.05 by repeated measures one-way ANOVA with Geisser-Greenhouse correction and Tukey post-test from n = 3 independent experiments. B-C , YO-PRO-1 fluorescence was measured in primary mouse peritoneal macrophages at baseline, followed by stimulation with 300 µM BzATP over the course of 20 min. The leftmost graph in each panel indicates the average fluorescence intensity of YO-PRO-1 within cells over time (minutes). The middle graph depicts representative single cell tracings of YO-PRO-1 dye uptake. The rightmost graph (violin plot) in each panel indicates average YO-PRO-1 fluorescence intensity within individual cells (circles, squares) at 20 minutes following BzATP treatment. Peritoneal macrophages were stimulated with or without P2X7R perturbation by genetic knockout ( P2rx7 KO) ( B ) or pre-treatment with A438079 ( C ). D-E , Intracellular calcium measurements in primary mouse peritoneal macrophages before and after the addition of 300 µM BzATP. Graph indicates the ratio of fluorescence intensity of Fura-2 at 340 and 380 nm over time following BzATP treatment. Timepoints are shown relative to the first recorded measurement following BzATP treatment (t = 0 s). Peritoneal macrophages were stimulated with or without P2X7R perturbation by genetic knockout ( P2rx7 KO) ( D ) or pre-treatment with A438079 ( E ).

Article Snippet: R&D-AF5105) and P2X7 antibody (1:1000, Novus-NBP1-20180).

Techniques: Fluorescence, Knock-Out

Journal: The Journal of general physiology

Article Title: Extracellular histone proteins activate P2XR7 channel current.

doi: 10.1085/jgp.202213317

Figure Lengend Snippet: Figure 1. Purified histone octamer proteins activate P2XR7 currents. (A) Representative traces of current versus time recorded from a Xenopus oocyte 24–48 h after nuclear injection of plasmid cDNA encoding mouse P2XR7. Cells were held at −60 mV. 1 s voltage ramps from −60 to 40 mV were performed at baseline and in the presence of 600 μM ATP or 10 μg/ml histone protein in high Ca2+ Ringer’s solution. (B and C) Average whole-cell current at −60 mV in oocytes 24–48 h after nuclear injection of mouse (B, “m.”) or human (C, “h.”) P2XR7 cDNA in response to 600 μM ATP, 50 μM BzATP, or 10 μg/ml histones (uninjected cells, “Ctrl”; Ave ± SEM, n = 3–9 animals, 8–28 cells). (D) ATP-, BzATP-, and histone-evoked m.P2XR7 current decay with agonist removal estimated by fitting data from A (highlighted in gray) to a single exponential (Ave ± SEM, n = 3 animals, 3–10 cells, * P = 0.0016 histone vs. BzATP, * P < 0.0001 histone vs. ATP). (E and F) Representative current–voltage relationships measured once peak sustained currents were achieved in A (n = 3 animals, 10 cells). (G and H) Average reversal potential (G) and whole-cell conductance (H) for ATP- and histone-evoked currents in oocytes expressing P2XR7 calculated from voltage ramps in E and F (Ave ± SEM, n = 3 animals, 10 cells). (I and J) average ATP- or histone-evoked inward current measured at different holding potentials (Ave ± SEM, some error bars are smaller than their marker, n = 2–3 animals, 8–10 cells, reversal = −24 mV for ATP and −39 mV for histone). Gray line represents best fit linear regression. (J) Histone-evoked currents were normalized to ATP-evoked currents from the same cell (Ave ± SEM, some error bars are smaller than their marker, n = 2–3 animals, 8–10 cells, reversal = −35 mV). (K) Average whole-cell current in P2XR7-expressing cells in response to histone concentrations from 1 to 10 μg/ml and in response to boiled and Pro K digested histone protein at 10 μg/ml (Pro K–treated histones also boiled to inactivate Pro K, Ave ± SEM, n = 3–9 animals, 8–20 cells, * P < 0.0001 by non-parametric one-way ANOVA).

Article Snippet: cDNA for mouse and human P2XR7 was obtained from Origene (MR227216 and RC222143; Origene Technologies).

Techniques: Purification, Injection, Plasmid Preparation, Expressing, Marker

Journal: The Journal of general physiology

Article Title: Extracellular histone proteins activate P2XR7 channel current.

doi: 10.1085/jgp.202213317

Figure Lengend Snippet: Figure 2. Histone-evoked P2XR7 currents are inhibited by non-selective P2XR antagonists, but not inhibited by selective P2XR7 antago- nists. (A) Representative trace of current versus time recorded from a Xenopus oocyte 24–48 h after nuclear injection of plasmid cDNA encoding mouse P2XR7. 5 μM Suramin inhibits ATP- and histone-evoked currents. (B) Average current after 30 s of antagonist treatment relative to baseline histone-evoked current (Ctrl, 30 s time/ vehicle control; 5 μM PPADS, 10 μM TNP-ATP, 5 μM Suramin, Ave ± SEM, n = 3–4 animals, 7–18 cells, P values from ANOVA with multiple com- parisons are reported in the figure). (C–H) Rep- resentative trace and average ATP-evoked current after application of indicated P2XR7- selective antagonists on ATP-activated, open, channels. (D) 10 μM AZ10606120 (AZ10, Ave ± SEM, n = 3 animals, 18 cells). (E) 10 μM A438079 (A43, Ave ± SEM, n = 2 animal, 8 cells). (F) 50 μM GW791343 (GW, Ave ± SEM, n = 5 animals, 16 cells). (G) 10 μM AZ11645373 (AZ11, Ave ± SEM, n = 3 animals, 8 cells). (H) 1 μM KN-62 (KN62, Ave ± SEM, n = 2 animals, 7 cells). P values by Student’s t test indicated above each graph. (I–N) Representative trace and average histone- evoked current after application of indicated P2XR7-selective antagonist on histone-activated, open, channels. (J) 10 μM AZ10606120 (AZ10, Ave ± SEM, n = 3 animals, 10 cells). (K) 10 μM A438079 (A43, Ave ± SEM, n = 2 animals, 8 cells). (L) 50 μM GW791343 (GW, Ave ± SEM, n = 2 animals, 9 cells). (M) 10 μM AZ11645373 (AZ11, Ave ± SEM, n = 3 animals, 8 cells). (N) 1 μM KN-62 (KN62, Ave± SEM, n= 1 animal, 3 cells). P values by Student’s t test indicated above each graph. (O–Q) Representative trace (O) and average ATP- and histone-evoked currents before (control) and after pre-application of P2XR7-selective antagonists. (P) ATP-evoked currents (Ave ± SEM) after pre- application of AZ10 (n = 2 animals, 9 cells), A43 (n = 2 animals, 8 cells), GW (n = 3 animals, 9 cells), AZ11 (n= 3 animals, 9 cells), or KN62 (n= 2 animals, 7 cells). (Q) Histone-evoked currents (Ave ± SEM) after pre-application of AZ10 (n = 3 animals, 15 cells), A43 (n = 3 animals, 11 cells), GW (n = 2 ani- mals, 9 cells), AZ11 (n = 2 animals, 7 cells), or KN62 (n = 2 animals, 9 cells). P values by one-way AN- OVA (non-parametric) indicated above the graph.

Article Snippet: cDNA for mouse and human P2XR7 was obtained from Origene (MR227216 and RC222143; Origene Technologies).

Techniques: Injection, Plasmid Preparation, Control

Journal: The Journal of general physiology

Article Title: Extracellular histone proteins activate P2XR7 channel current.

doi: 10.1085/jgp.202213317

Figure Lengend Snippet: Figure 4. Mouse P2XR7 variants, 451L and -P, are activated by ATP and histones. (A) Average ATP-evoked current as a function of ATP concentration from cells expressing mouse P2XR7 451P and 451L variants (Ave ± SEM, n = 4–7 animals, 13–21 cells). (B) Average ATP-evoked (500 μM) currents for 451P and 451L variants (Ave ± SEM, n = 4–7 animals, 20–21 cells, P = 0.0482). (C) Average histone-evoked (20 μg/ml) currents for 451P and 451L variants (Ave ± SEM, n = 4–7 animals, 20–21 cells, P = 0.2709).

Article Snippet: cDNA for mouse and human P2XR7 was obtained from Origene (MR227216 and RC222143; Origene Technologies).

Techniques: Concentration Assay, Expressing

Journal: The Journal of general physiology

Article Title: Extracellular histone proteins activate P2XR7 channel current.

doi: 10.1085/jgp.202213317

Figure Lengend Snippet: Figure 3. Histone-evoked P2XR7 currents are increased in low Ca2+

Article Snippet: cDNA for mouse and human P2XR7 was obtained from Origene (MR227216 and RC222143; Origene Technologies).

Techniques:

Journal: The Journal of general physiology

Article Title: Extracellular histone proteins activate P2XR7 channel current.

doi: 10.1085/jgp.202213317

Figure Lengend Snippet: Figure 5. Histone H2b, H3, and H4, but not H2a, activate P2XR7 currents. (A and B) Representative trace of P2XR7 currents at −60 mV activated by purified recombinant human H2b (A), H3 (B, top), and H4 (B, bottom; 1 μg/ml, each). (C) Average histone-evoked current at −60 mV in control (Ctrl) and mouse P2XR7 (451L) expressing oocytes (n = 2–3 animals, 6–17 cells, P = 0.291 (H2a), P = 0.0002 (H2b), P = < 0.0001 (H3), P < 0.0001 (H4) by unpaired Student’s t test (P2XR7 expressing versus control).

Article Snippet: cDNA for mouse and human P2XR7 was obtained from Origene (MR227216 and RC222143; Origene Technologies).

Techniques: Purification, Recombinant, Control, Expressing

Journal: International journal of medical microbiology : IJMM

Article Title: Chlamydia psittaci inhibits apoptosis of human neutrophils by activating P2X7 receptor expression.

doi: 10.1016/j.ijmm.2022.151571

Figure Lengend Snippet: Fig. 5. The role of the P2X7 receptor in hPMN apoptosis. (A) C. psittaci 6 BCE infected hPMN for 24 h and then cells were probed with rabbit anti-P2X7 mAb and Wheat Germ Agglutinin (WGA), which stain in red, the second antibody was Cy2-conjugated goat anti-rabbit IgG (green), then stain with DAPI (blue). bar= 20 µm. (B) Whole-cell lysates were collected from hPMN at 24 h. Western Blots showed the expression of P2X7 and β-actin was used as a loading control. The data shown are from one experiment that is representative of three independent determinations. (C) At 24 h, hPMN were infected with C. psittaci. Before infection, hPMN were incubated for 1 h with 10 nM AZ10606120 or 100 μM BzATP. (D) Graphs represent the percentages of viable cells as PI and annexin-V double-negative cells. (E) HPMN were incubated for 1 h with 100 nM Z-DEVD-FMK for 24 h, caspase-3 activities were measured using luminescence assays. RLU, Relative light units. Data are the mean ± SEM of triplicate samples from a representative experiment. *P < 0.05, **P < 0.01. ***P < 0.001.

Article Snippet: Rabbit anti-P2X7 polyclonal antibodies were obtained from ORIGENE (Rockville, Maryland, USA) and mouse anti-β-actin mAb, rabbit anti-IgG and mouse anti-IgG were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Infection, Staining, Western Blot, Expressing, Control, Incubation