rabbit anti panx1 antibody  (Alomone Labs)


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    Structured Review

    Alomone Labs rabbit anti panx1 antibody
    Cholesterol depletion reduces lateral diffusion of mPanx-1-GFP in <t>Panx1-null</t> astrocytes. ( A ) <t>Panx1-null</t> astrocytes transiently expressing mPanx-1-GFP were subjected to treatments with 5 mM methyl-β-cyclodextrin (MβCD), 5 mM MβCD followed by addition of cholesterol (MβCD + Chol) or left untreated (CTRL). Typical FRAP curves of the lateral diffusion of mPanx1-GFP and its modulation by the various treatments are depicted in A. mPanx1-GFP lateral diffusion rates were reduced by MβCD ( B ). Normal diffusion was restored when cholesterol was added. (* p
    Rabbit Anti Panx1 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 94 stars, based on 13 article reviews
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    rabbit anti panx1 antibody - by Bioz Stars, 2022-11
    94/100 stars

    Images

    1) Product Images from "Activity and Stability of Panx1 Channels in Astrocytes and Neuroblastoma Cells Are Enhanced by Cholesterol Depletion"

    Article Title: Activity and Stability of Panx1 Channels in Astrocytes and Neuroblastoma Cells Are Enhanced by Cholesterol Depletion

    Journal: Cells

    doi: 10.3390/cells11203219

    Cholesterol depletion reduces lateral diffusion of mPanx-1-GFP in Panx1-null astrocytes. ( A ) Panx1-null astrocytes transiently expressing mPanx-1-GFP were subjected to treatments with 5 mM methyl-β-cyclodextrin (MβCD), 5 mM MβCD followed by addition of cholesterol (MβCD + Chol) or left untreated (CTRL). Typical FRAP curves of the lateral diffusion of mPanx1-GFP and its modulation by the various treatments are depicted in A. mPanx1-GFP lateral diffusion rates were reduced by MβCD ( B ). Normal diffusion was restored when cholesterol was added. (* p
    Figure Legend Snippet: Cholesterol depletion reduces lateral diffusion of mPanx-1-GFP in Panx1-null astrocytes. ( A ) Panx1-null astrocytes transiently expressing mPanx-1-GFP were subjected to treatments with 5 mM methyl-β-cyclodextrin (MβCD), 5 mM MβCD followed by addition of cholesterol (MβCD + Chol) or left untreated (CTRL). Typical FRAP curves of the lateral diffusion of mPanx1-GFP and its modulation by the various treatments are depicted in A. mPanx1-GFP lateral diffusion rates were reduced by MβCD ( B ). Normal diffusion was restored when cholesterol was added. (* p

    Techniques Used: Diffusion-based Assay, Expressing

    Human Panx1 conductance is activated by cholesterol depletion. ( A , B ) Electrophysiological recordings obtained from N2a cells expressing hPanx1 subjected to treatment with 5 mM MβCD ( A , D ) and 5 µM lovastatin ( B , E ). Note that N2a hPanx-1 transfected cells responded to cholesterol depletion with increased current amplitudes (red and green curves, in A and B, respectively) in response to 5.5 s-long voltage ramps from −60 to +80 mV compared to untreated cells, CTRL (black curves). Mean ± SEM values of the fold changes in peak conductance induced by 5 mM MβCD and 10 µM lovastatin obtained for N2a hPanx1 transfected cells are shown in parts ( C , D ), respectively. p values were obtained using Mann–Whitney tests. n = 10–11 cells. ** p
    Figure Legend Snippet: Human Panx1 conductance is activated by cholesterol depletion. ( A , B ) Electrophysiological recordings obtained from N2a cells expressing hPanx1 subjected to treatment with 5 mM MβCD ( A , D ) and 5 µM lovastatin ( B , E ). Note that N2a hPanx-1 transfected cells responded to cholesterol depletion with increased current amplitudes (red and green curves, in A and B, respectively) in response to 5.5 s-long voltage ramps from −60 to +80 mV compared to untreated cells, CTRL (black curves). Mean ± SEM values of the fold changes in peak conductance induced by 5 mM MβCD and 10 µM lovastatin obtained for N2a hPanx1 transfected cells are shown in parts ( C , D ), respectively. p values were obtained using Mann–Whitney tests. n = 10–11 cells. ** p

    Techniques Used: Expressing, Transfection, MANN-WHITNEY

    Panx1 channels mediate dye uptake in cholesterol-depleted cell plasma membrane of N2a cells. ( A ) Representative photomicrographs of mPanx1 transfected and Panx1-null. ( C ) N2a cells showing EthBr uptake after MβCD and lovastatin with and without 100 µM BzATP treatment. Scale bar: 100 µm. ( B , D ) Mean ± SEM values of the relative EthBr fluorescence intensity. Values were normalized to those obtained under control conditions. Each point on the graphs represents the mean values of relative EthBr fluorescence changes recorded from all cells present in a field of view obtained from 3–5 independent experiments. p values obtained from ANOVA followed by Dunnett’s test. * p
    Figure Legend Snippet: Panx1 channels mediate dye uptake in cholesterol-depleted cell plasma membrane of N2a cells. ( A ) Representative photomicrographs of mPanx1 transfected and Panx1-null. ( C ) N2a cells showing EthBr uptake after MβCD and lovastatin with and without 100 µM BzATP treatment. Scale bar: 100 µm. ( B , D ) Mean ± SEM values of the relative EthBr fluorescence intensity. Values were normalized to those obtained under control conditions. Each point on the graphs represents the mean values of relative EthBr fluorescence changes recorded from all cells present in a field of view obtained from 3–5 independent experiments. p values obtained from ANOVA followed by Dunnett’s test. * p

    Techniques Used: Transfection, Fluorescence

    Cholesterol depletion enhances dye uptake through Panx1 channels in astrocytes. ( A ) Fluorescence micrographs of intracellular EthBr in normal conditions (CTRL) and after treatment with MβCD, BzATP, or with both drugs. Scale bar: 100 µm. ( B ) EthBr uptake in Panx1-null astrocytes induced by MβCD with and without BzATP. Scale bar: 100 µm. ( C ) Histograms showing relative EthBr uptake compared to controls for treatment with MβCD alone or together with BzATP. ( D ) Mean ± SEM values of the relative EthBr fluorescence intensity obtained from Panx1-null astrocytes after MβCD, 100 µM BzATP, and MβCD with BzATP treatment. Values were normalized to those obtained under control conditions. In ( C , D ), each bar on the graphs represents mean value of relative EthBr fluorescence changes recorded from all cells present in a field of view obtained from 3–5 independent experiments. p values obtained from ANOVA test followed by Dunnett’s test. *** p
    Figure Legend Snippet: Cholesterol depletion enhances dye uptake through Panx1 channels in astrocytes. ( A ) Fluorescence micrographs of intracellular EthBr in normal conditions (CTRL) and after treatment with MβCD, BzATP, or with both drugs. Scale bar: 100 µm. ( B ) EthBr uptake in Panx1-null astrocytes induced by MβCD with and without BzATP. Scale bar: 100 µm. ( C ) Histograms showing relative EthBr uptake compared to controls for treatment with MβCD alone or together with BzATP. ( D ) Mean ± SEM values of the relative EthBr fluorescence intensity obtained from Panx1-null astrocytes after MβCD, 100 µM BzATP, and MβCD with BzATP treatment. Values were normalized to those obtained under control conditions. In ( C , D ), each bar on the graphs represents mean value of relative EthBr fluorescence changes recorded from all cells present in a field of view obtained from 3–5 independent experiments. p values obtained from ANOVA test followed by Dunnett’s test. *** p

    Techniques Used: Fluorescence

    Panx1 channels mediate ATP release from cultured cholesterol depleted astrocytes. Histograms showing the ATP release from cultured Panx1 f/f and Panx1-null astrocytes induced by cholesterol depletion with 5 mM MβCD ( A , B ) and 10 µM lovastatin ( C , D ). Bar histogram of the mean ± SEM values of ATP release from cultured Panx1 f/f and Panx1 null astrocytes induced by cholesterol depletion. p values were obtained using Mann–Whitney tests. Data are from 2–3 independent experiments. * p
    Figure Legend Snippet: Panx1 channels mediate ATP release from cultured cholesterol depleted astrocytes. Histograms showing the ATP release from cultured Panx1 f/f and Panx1-null astrocytes induced by cholesterol depletion with 5 mM MβCD ( A , B ) and 10 µM lovastatin ( C , D ). Bar histogram of the mean ± SEM values of ATP release from cultured Panx1 f/f and Panx1 null astrocytes induced by cholesterol depletion. p values were obtained using Mann–Whitney tests. Data are from 2–3 independent experiments. * p

    Techniques Used: Cell Culture, MANN-WHITNEY

    Effects of treatment with MβCD and lovastatin on membrane cholesterol level, Panx1 extraction, and cell viability. ( A ) mPanx-1-GFP transfected N2a cells were incubated with cyclodextrin (5 mM MβCD and 10 mM MβCD for 60 min at 37 °C), lovastatin (5 μM, 12 h) in serum-free DMEM or with cholesterol (2 h, 37 °C). Washed membrane fractions were assayed for cholesterol with results normalized with respect to the protein content of the membrane fraction. Bars show mean ± SEM of replicates of three experiments. ( B ) Effects of MβCD and lovastatin in medium of Panx1 cells. ( C ) Effects of MβCD, lovastatin, and cholesterol on cell viability of mPanx1-GFP transfected N2a cells measured by MTT assay after treatments. p values obtained from ANOVA test followed by Dunnett’s test. Results are representative of three independent experiments ± SEM.* p
    Figure Legend Snippet: Effects of treatment with MβCD and lovastatin on membrane cholesterol level, Panx1 extraction, and cell viability. ( A ) mPanx-1-GFP transfected N2a cells were incubated with cyclodextrin (5 mM MβCD and 10 mM MβCD for 60 min at 37 °C), lovastatin (5 μM, 12 h) in serum-free DMEM or with cholesterol (2 h, 37 °C). Washed membrane fractions were assayed for cholesterol with results normalized with respect to the protein content of the membrane fraction. Bars show mean ± SEM of replicates of three experiments. ( B ) Effects of MβCD and lovastatin in medium of Panx1 cells. ( C ) Effects of MβCD, lovastatin, and cholesterol on cell viability of mPanx1-GFP transfected N2a cells measured by MTT assay after treatments. p values obtained from ANOVA test followed by Dunnett’s test. Results are representative of three independent experiments ± SEM.* p

    Techniques Used: Transfection, Incubation, MTT Assay

    Cholesterol depletion reduces lateral mobility of Panx1 in the membrane. N2a cells transiently expressing mPanx-1-GFP were subjected to treatment with 5 mM methyl-β-cyclodextrin (MβCD), 5 mM MβCD followed by addition of cholesterol (MβCD + Chol) or left untreated (CTRL). ( A – C ) Fluorescence images of transfected cells (control and treated with MβCD alone or with cholesterol) before (pre-bleach) and at 0.6 and 60 s postbleach; bleached regions are shown at higher magnification in insets. Bars: 10 μm; inset magnification 3×. ( D ) FRAP recovery curves for control cells and with MβCD alone or with cholesterol. ( E ) Histograms of normalized FRAP recovery at 15, 30, and 60 s after photobleach. p values obtained from ANOVA test followed by Dunnett’s test. n = 7–10 per plasma membrane domain, data collected over four independent repeats. ** p
    Figure Legend Snippet: Cholesterol depletion reduces lateral mobility of Panx1 in the membrane. N2a cells transiently expressing mPanx-1-GFP were subjected to treatment with 5 mM methyl-β-cyclodextrin (MβCD), 5 mM MβCD followed by addition of cholesterol (MβCD + Chol) or left untreated (CTRL). ( A – C ) Fluorescence images of transfected cells (control and treated with MβCD alone or with cholesterol) before (pre-bleach) and at 0.6 and 60 s postbleach; bleached regions are shown at higher magnification in insets. Bars: 10 μm; inset magnification 3×. ( D ) FRAP recovery curves for control cells and with MβCD alone or with cholesterol. ( E ) Histograms of normalized FRAP recovery at 15, 30, and 60 s after photobleach. p values obtained from ANOVA test followed by Dunnett’s test. n = 7–10 per plasma membrane domain, data collected over four independent repeats. ** p

    Techniques Used: Expressing, Fluorescence, Transfection

    Expression of endogenous Panx1 is largely absent in CRISPR-deleted N2a cells. Representative confocal images obtained from ( A ) parental, ( B ) Panx1-deleted, and ( C ) Panx1-deleted N2a cells expressing human (h) Panx1 construct. Boxed white areas show the Panx1 staining at higher magnification. Scale bars: 50 μm lower magnification, 20 μm in boxed areas.
    Figure Legend Snippet: Expression of endogenous Panx1 is largely absent in CRISPR-deleted N2a cells. Representative confocal images obtained from ( A ) parental, ( B ) Panx1-deleted, and ( C ) Panx1-deleted N2a cells expressing human (h) Panx1 construct. Boxed white areas show the Panx1 staining at higher magnification. Scale bars: 50 μm lower magnification, 20 μm in boxed areas.

    Techniques Used: Expressing, CRISPR, Construct, Staining

    2) Product Images from "Mechanosensitive Pannexin 1 Activity Is Modulated by Stomatin in Human Red Blood Cells"

    Article Title: Mechanosensitive Pannexin 1 Activity Is Modulated by Stomatin in Human Red Blood Cells

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms23169401

    TO-PRO-3 uptake in viable and non-apoptotic stom + and stom − K562 cells after hypotonic stress. ( A ) Expression of PANX1 in stom − and stom + K562 clones was assessed by flow cytometry, giving similar levels. ( B ) Comparison of the relative TO-PRO-3 fluorescence intensity (F − F0/F0 − 100) between stom + (black squares) and stom − (grey squares) K562 cell clones using an unpaired t-test. ( C ) Cell volume of stom + (black) and stom − (grey) K562 clones assessed by flow cytometry comparing FSC geomean values obtained in isotonic (circles) and hypotonic (80 mosm/kg, squares) conditions. ( D ) Caspase 3/7 activity measured by flow cytometry analysis allowing the determination of the percentage of fluorescent stom + (iso: black and hypo: black with white dots) and stom − (iso: grey and hypo: grey with white dots) K562 cell clones preloaded by the NucView substrate and stimulated by hypotonic stress (two-way ANOVA Sidak’s multiple comparisons test). ( p values > 0.05: ns, ≤0.05: *, ≤0.01: ** and ≤0.0001: ****).
    Figure Legend Snippet: TO-PRO-3 uptake in viable and non-apoptotic stom + and stom − K562 cells after hypotonic stress. ( A ) Expression of PANX1 in stom − and stom + K562 clones was assessed by flow cytometry, giving similar levels. ( B ) Comparison of the relative TO-PRO-3 fluorescence intensity (F − F0/F0 − 100) between stom + (black squares) and stom − (grey squares) K562 cell clones using an unpaired t-test. ( C ) Cell volume of stom + (black) and stom − (grey) K562 clones assessed by flow cytometry comparing FSC geomean values obtained in isotonic (circles) and hypotonic (80 mosm/kg, squares) conditions. ( D ) Caspase 3/7 activity measured by flow cytometry analysis allowing the determination of the percentage of fluorescent stom + (iso: black and hypo: black with white dots) and stom − (iso: grey and hypo: grey with white dots) K562 cell clones preloaded by the NucView substrate and stimulated by hypotonic stress (two-way ANOVA Sidak’s multiple comparisons test). ( p values > 0.05: ns, ≤0.05: *, ≤0.01: ** and ≤0.0001: ****).

    Techniques Used: Expressing, Clone Assay, Flow Cytometry, Fluorescence, Activity Assay

    Endogenous expression of stomatin and PANX1 in K562 cells and PANX1-dependent TO-PRO-3 uptake by non-apoptotic K562 cells. ( A ) Stomatin expression analysed by flow cytometry on permeabilized K562 cells using anti-stomatin E5 (Santa Cruz Biotechnology, Dallas, TX, USA). ( B ) PANX1 expression analysed by flow cytometry on permeabilized K562 cells using anti-PANX1 (Alomone Labs, Jerusalem, Israel). ( C ) Gating strategy of Sytox-negative (Pacific Blue) and Lactadherin-negative (FITC) K562 cells (at least 85%, Q4) in which the TO-PRO-3 staining (APC) was evaluated by flow cytometry. ( D ) TO-PRO-3 uptake was measured on K562 cells in basal conditions and, after a hypotonic stimulation (80 mOsm/kg), on K562 cells that were preincubated or not with several PANX1 inhibitors (carbenoxolone [CBX], probenecid [PBC], and mefloquine [MFQ]) (N = 3).
    Figure Legend Snippet: Endogenous expression of stomatin and PANX1 in K562 cells and PANX1-dependent TO-PRO-3 uptake by non-apoptotic K562 cells. ( A ) Stomatin expression analysed by flow cytometry on permeabilized K562 cells using anti-stomatin E5 (Santa Cruz Biotechnology, Dallas, TX, USA). ( B ) PANX1 expression analysed by flow cytometry on permeabilized K562 cells using anti-PANX1 (Alomone Labs, Jerusalem, Israel). ( C ) Gating strategy of Sytox-negative (Pacific Blue) and Lactadherin-negative (FITC) K562 cells (at least 85%, Q4) in which the TO-PRO-3 staining (APC) was evaluated by flow cytometry. ( D ) TO-PRO-3 uptake was measured on K562 cells in basal conditions and, after a hypotonic stimulation (80 mOsm/kg), on K562 cells that were preincubated or not with several PANX1 inhibitors (carbenoxolone [CBX], probenecid [PBC], and mefloquine [MFQ]) (N = 3).

    Techniques Used: Expressing, Flow Cytometry, Staining

    Activation of ATP release in frozen/thawed and rejuvenated RBCs from controls and OHSt patients. Suspensions of frozen/thawed and rejuvenated RBCs were exposed to isoosmotic (Iso) or hypoosmotic (Hypo, 240 mOsm/kg) stimulation. Experiments were performed with or without preincubation with 100 μM CBX, a PANX1 inhibitor. Results are expressed as eATP (pmol/10 6 cells). ( A ) Stacked graph showing total eATP including lytic eATP (hatched bars) released from control (white bars) or OHSt (grey bars) RBCs. The hemoglobin concentration of paired samples was used to estimate lytic eATP. ( B ) Non-lytic eATP was calculated as the differences between total eATP and lytic eATP. Total, lytic, and non-lytic eATP values were compared using a One-Way ANOVA Dunnett’s test for Multiple Comparison (N = 3). The results shown are mean values ± SEM. ( p values > 0.05: ns, ≤0.05: *, ≤0.01: **). ns stands for not significant.
    Figure Legend Snippet: Activation of ATP release in frozen/thawed and rejuvenated RBCs from controls and OHSt patients. Suspensions of frozen/thawed and rejuvenated RBCs were exposed to isoosmotic (Iso) or hypoosmotic (Hypo, 240 mOsm/kg) stimulation. Experiments were performed with or without preincubation with 100 μM CBX, a PANX1 inhibitor. Results are expressed as eATP (pmol/10 6 cells). ( A ) Stacked graph showing total eATP including lytic eATP (hatched bars) released from control (white bars) or OHSt (grey bars) RBCs. The hemoglobin concentration of paired samples was used to estimate lytic eATP. ( B ) Non-lytic eATP was calculated as the differences between total eATP and lytic eATP. Total, lytic, and non-lytic eATP values were compared using a One-Way ANOVA Dunnett’s test for Multiple Comparison (N = 3). The results shown are mean values ± SEM. ( p values > 0.05: ns, ≤0.05: *, ≤0.01: **). ns stands for not significant.

    Techniques Used: Activation Assay, Concentration Assay

    5(6)-carboxyfluorescein uptake in control (Ctrl) and OHSt RBCs after stimulation by high K + . ( A ) MFI due to the dye uptake in Ctrl (black) and OHSt (grey) frozen/thawed rejuvenated RBCs measured by flow cytometry in basal conditions and after stimulation by KCl without or with incubation with CBX. ( B ) Carbenoxolone (CBX), probenecid (PBC), and mefloquine (MFQ), three different PANX1 inhibitors, were added to RBC 20 min before the stimulation, resulting in a fluorescence signal similar to that of basal conditions. ( C ) FSC was also determined in all the conditions described above under KCl stimulation. Data obtained from stimulated cells were compared to that of unstimulated cells or cells stimulated in the presence of CBX using a paired Two-way Anova Sidak’s multiple comparisons test. The same test but unpaired was used when Ctrl and OHSt RBCs were compared. ( p values > 0.05: ns, ≤0.05: *, ≤0.0001: ****). ns stands for not significant.
    Figure Legend Snippet: 5(6)-carboxyfluorescein uptake in control (Ctrl) and OHSt RBCs after stimulation by high K + . ( A ) MFI due to the dye uptake in Ctrl (black) and OHSt (grey) frozen/thawed rejuvenated RBCs measured by flow cytometry in basal conditions and after stimulation by KCl without or with incubation with CBX. ( B ) Carbenoxolone (CBX), probenecid (PBC), and mefloquine (MFQ), three different PANX1 inhibitors, were added to RBC 20 min before the stimulation, resulting in a fluorescence signal similar to that of basal conditions. ( C ) FSC was also determined in all the conditions described above under KCl stimulation. Data obtained from stimulated cells were compared to that of unstimulated cells or cells stimulated in the presence of CBX using a paired Two-way Anova Sidak’s multiple comparisons test. The same test but unpaired was used when Ctrl and OHSt RBCs were compared. ( p values > 0.05: ns, ≤0.05: *, ≤0.0001: ****). ns stands for not significant.

    Techniques Used: Flow Cytometry, Incubation, Fluorescence

    Activation of ATP release in fresh RBCs by a hypoosmotic challenge. ( A ) RBC suspensions were exposed to isoosmotic (Iso) or hypoosmotic (Hypo, 240 mOsm/kg) stimulation. Experiments were performed with or without preincubation with 100 μM CBX, a PANX1 inhibitor. Results are expressed as eATP (pmol/10 6 cells). The hemoglobin concentration of paired samples was used to estimate lytic eATP. A stacked graph was built to show the contribution of lytic ATP (hatched bars) within total ATP release from RBCs. Lytic and total eATP values were compared using a One-Way ANOVA Dunnett’s test for Multiple Comparisons (N = 5). The results shown are mean values ± SEM. ( B ) The difference between total eATP and lytic eATP was calculated and denoted as non-lytic eATP. Non-lytic eATP values were compared using a One-Way ANOVA Dunnett’s test for Multiple Comparisons. ( C ) PS exposure was measured by flow cytometry on Lactadherin-FITC stained RBCs in basal conditions after treatment with Fenton’s reagent, as a control of eryptosis, and in basal or hypoosmotic conditions without Fenton. ( D ) Caspase activation was measured under conditions similar to PS exposure but on FLICA 660 caspase3/7-stained RBCs. The statistical analysis of the last two measurements (N = 5) was performed using an RM-one-way ANOVA test. ( p values > 0.05: ns, ≤0.05: *, ≤0.01: ** and ≤0.001: ***). ns stands for not significant.
    Figure Legend Snippet: Activation of ATP release in fresh RBCs by a hypoosmotic challenge. ( A ) RBC suspensions were exposed to isoosmotic (Iso) or hypoosmotic (Hypo, 240 mOsm/kg) stimulation. Experiments were performed with or without preincubation with 100 μM CBX, a PANX1 inhibitor. Results are expressed as eATP (pmol/10 6 cells). The hemoglobin concentration of paired samples was used to estimate lytic eATP. A stacked graph was built to show the contribution of lytic ATP (hatched bars) within total ATP release from RBCs. Lytic and total eATP values were compared using a One-Way ANOVA Dunnett’s test for Multiple Comparisons (N = 5). The results shown are mean values ± SEM. ( B ) The difference between total eATP and lytic eATP was calculated and denoted as non-lytic eATP. Non-lytic eATP values were compared using a One-Way ANOVA Dunnett’s test for Multiple Comparisons. ( C ) PS exposure was measured by flow cytometry on Lactadherin-FITC stained RBCs in basal conditions after treatment with Fenton’s reagent, as a control of eryptosis, and in basal or hypoosmotic conditions without Fenton. ( D ) Caspase activation was measured under conditions similar to PS exposure but on FLICA 660 caspase3/7-stained RBCs. The statistical analysis of the last two measurements (N = 5) was performed using an RM-one-way ANOVA test. ( p values > 0.05: ns, ≤0.05: *, ≤0.01: ** and ≤0.001: ***). ns stands for not significant.

    Techniques Used: Activation Assay, Concentration Assay, Flow Cytometry, Staining

    Stomatin and PANX1 proximity in control and OHSt RBCs assessed by Proximity Ligation Assay (PLA) and analyzed by Imaging Flow Cytometry. ( A ) PLA-positive (PLA+) events characterized by red spots which result from the detection oligos coupled to fluorochromes (FarRed) hybridizing to sequences in the amplified DNA were visualized by Imaging Flow Cytometry and merged with Brightfield images of the corresponding Control RBCs. ( B ) Quantification of the assay corresponding to the percentage of PLA+ RBCs was performed several times in different conditions: without any primary antibody (none, N = 10), with only one antibody (anti-stomatin or anti-PANX1 or anti-AE1, N = 3 for each condition) and in the presence of two antibodies (anti-stomatin + anti-PANX1 or anti-stomatin + anti-AE1, N = 7 in both conditions). A comparison was performed using an unpaired One-way Anova Tukey’s test. ( C ) PLA using anti-stomatin + anti-PANX1 was performed in identical conditions to those described above but on frozen/thawed control (Ctrl) and OHSt RBCs and compared using paired or unpaired (between control and OHSt) Two-way Anova Sidak’s multiple comparisons test ( p values > 0.05: ns, ≤0.05: *, ≤0.01: ** and ≤0.001: ***). ns stands for not significant.
    Figure Legend Snippet: Stomatin and PANX1 proximity in control and OHSt RBCs assessed by Proximity Ligation Assay (PLA) and analyzed by Imaging Flow Cytometry. ( A ) PLA-positive (PLA+) events characterized by red spots which result from the detection oligos coupled to fluorochromes (FarRed) hybridizing to sequences in the amplified DNA were visualized by Imaging Flow Cytometry and merged with Brightfield images of the corresponding Control RBCs. ( B ) Quantification of the assay corresponding to the percentage of PLA+ RBCs was performed several times in different conditions: without any primary antibody (none, N = 10), with only one antibody (anti-stomatin or anti-PANX1 or anti-AE1, N = 3 for each condition) and in the presence of two antibodies (anti-stomatin + anti-PANX1 or anti-stomatin + anti-AE1, N = 7 in both conditions). A comparison was performed using an unpaired One-way Anova Tukey’s test. ( C ) PLA using anti-stomatin + anti-PANX1 was performed in identical conditions to those described above but on frozen/thawed control (Ctrl) and OHSt RBCs and compared using paired or unpaired (between control and OHSt) Two-way Anova Sidak’s multiple comparisons test ( p values > 0.05: ns, ≤0.05: *, ≤0.01: ** and ≤0.001: ***). ns stands for not significant.

    Techniques Used: Proximity Ligation Assay, Imaging, Flow Cytometry, Amplification

    Stomatin and PANX1 expression in OHSt RBCs compared to controls. ( A ) Western blot analysis of RBC ghosts from 3 controls (C1, C2, C2) and the 3 OHSt patients (O1, O2, O3) using mouse monoclonal anti-stomatin (E-5), rabbit polyclonal anti-PANX1 and rabbit polyclonal anti-p55 as control. The molecular weights (kDa) of the detected bands are mentioned. ( B ) Flow cytometry analysis of stomatin expression in the RBCs of the 3 controls and the 3 OHSt patients after permeabilisation. Negative controls consist of the observed fluorescence with only the secondary antibody. The averages of Mean of Fluorescence Intensity (MFI) were compared between OHSt and control (Ctrl) RBCs using a two-way ANOVA Sidak’s multicomparison test ( p values > 0.05: ns and ≤0.05: *, ns stands for not significant), paired for comparisons within OHSt samples when signals with anti-stomatin were compared to that of Negative controls and unpaired when OHSt were compared to Ctrl. ( C ) Flow cytometry analysis of PANX1 expression in the 3 controls and the 3 OHSt patient RBCs after permeabilization and comparisons of MFIs between controls and OHSt, as performed in B.
    Figure Legend Snippet: Stomatin and PANX1 expression in OHSt RBCs compared to controls. ( A ) Western blot analysis of RBC ghosts from 3 controls (C1, C2, C2) and the 3 OHSt patients (O1, O2, O3) using mouse monoclonal anti-stomatin (E-5), rabbit polyclonal anti-PANX1 and rabbit polyclonal anti-p55 as control. The molecular weights (kDa) of the detected bands are mentioned. ( B ) Flow cytometry analysis of stomatin expression in the RBCs of the 3 controls and the 3 OHSt patients after permeabilisation. Negative controls consist of the observed fluorescence with only the secondary antibody. The averages of Mean of Fluorescence Intensity (MFI) were compared between OHSt and control (Ctrl) RBCs using a two-way ANOVA Sidak’s multicomparison test ( p values > 0.05: ns and ≤0.05: *, ns stands for not significant), paired for comparisons within OHSt samples when signals with anti-stomatin were compared to that of Negative controls and unpaired when OHSt were compared to Ctrl. ( C ) Flow cytometry analysis of PANX1 expression in the 3 controls and the 3 OHSt patient RBCs after permeabilization and comparisons of MFIs between controls and OHSt, as performed in B.

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

    3) Product Images from "Mechanosensitive Pannexin 1 Activity Is Modulated by Stomatin in Human Red Blood Cells"

    Article Title: Mechanosensitive Pannexin 1 Activity Is Modulated by Stomatin in Human Red Blood Cells

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms23169401

    TO-PRO-3 uptake in viable and non-apoptotic stom + and stom − K562 cells after hypotonic stress. ( A ) Expression of PANX1 in stom − and stom + K562 clones was assessed by flow cytometry, giving similar levels. ( B ) Comparison of the relative TO-PRO-3 fluorescence intensity (F − F0/F0 − 100) between stom + (black squares) and stom − (grey squares) K562 cell clones using an unpaired t-test. ( C ) Cell volume of stom + (black) and stom − (grey) K562 clones assessed by flow cytometry comparing FSC geomean values obtained in isotonic (circles) and hypotonic (80 mosm/kg, squares) conditions. ( D ) Caspase 3/7 activity measured by flow cytometry analysis allowing the determination of the percentage of fluorescent stom + (iso: black and hypo: black with white dots) and stom − (iso: grey and hypo: grey with white dots) K562 cell clones preloaded by the NucView substrate and stimulated by hypotonic stress (two-way ANOVA Sidak’s multiple comparisons test). ( p values > 0.05: ns, ≤0.05: *, ≤0.01: ** and ≤0.0001: ****).
    Figure Legend Snippet: TO-PRO-3 uptake in viable and non-apoptotic stom + and stom − K562 cells after hypotonic stress. ( A ) Expression of PANX1 in stom − and stom + K562 clones was assessed by flow cytometry, giving similar levels. ( B ) Comparison of the relative TO-PRO-3 fluorescence intensity (F − F0/F0 − 100) between stom + (black squares) and stom − (grey squares) K562 cell clones using an unpaired t-test. ( C ) Cell volume of stom + (black) and stom − (grey) K562 clones assessed by flow cytometry comparing FSC geomean values obtained in isotonic (circles) and hypotonic (80 mosm/kg, squares) conditions. ( D ) Caspase 3/7 activity measured by flow cytometry analysis allowing the determination of the percentage of fluorescent stom + (iso: black and hypo: black with white dots) and stom − (iso: grey and hypo: grey with white dots) K562 cell clones preloaded by the NucView substrate and stimulated by hypotonic stress (two-way ANOVA Sidak’s multiple comparisons test). ( p values > 0.05: ns, ≤0.05: *, ≤0.01: ** and ≤0.0001: ****).

    Techniques Used: Expressing, Clone Assay, Flow Cytometry, Fluorescence, Activity Assay

    Endogenous expression of stomatin and PANX1 in K562 cells and PANX1-dependent TO-PRO-3 uptake by non-apoptotic K562 cells. ( A ) Stomatin expression analysed by flow cytometry on permeabilized K562 cells using anti-stomatin E5 (Santa Cruz Biotechnology, Dallas, TX, USA). ( B ) PANX1 expression analysed by flow cytometry on permeabilized K562 cells using anti-PANX1 (Alomone Labs, Jerusalem, Israel). ( C ) Gating strategy of Sytox-negative (Pacific Blue) and Lactadherin-negative (FITC) K562 cells (at least 85%, Q4) in which the TO-PRO-3 staining (APC) was evaluated by flow cytometry. ( D ) TO-PRO-3 uptake was measured on K562 cells in basal conditions and, after a hypotonic stimulation (80 mOsm/kg), on K562 cells that were preincubated or not with several PANX1 inhibitors (carbenoxolone [CBX], probenecid [PBC], and mefloquine [MFQ]) (N = 3).
    Figure Legend Snippet: Endogenous expression of stomatin and PANX1 in K562 cells and PANX1-dependent TO-PRO-3 uptake by non-apoptotic K562 cells. ( A ) Stomatin expression analysed by flow cytometry on permeabilized K562 cells using anti-stomatin E5 (Santa Cruz Biotechnology, Dallas, TX, USA). ( B ) PANX1 expression analysed by flow cytometry on permeabilized K562 cells using anti-PANX1 (Alomone Labs, Jerusalem, Israel). ( C ) Gating strategy of Sytox-negative (Pacific Blue) and Lactadherin-negative (FITC) K562 cells (at least 85%, Q4) in which the TO-PRO-3 staining (APC) was evaluated by flow cytometry. ( D ) TO-PRO-3 uptake was measured on K562 cells in basal conditions and, after a hypotonic stimulation (80 mOsm/kg), on K562 cells that were preincubated or not with several PANX1 inhibitors (carbenoxolone [CBX], probenecid [PBC], and mefloquine [MFQ]) (N = 3).

    Techniques Used: Expressing, Flow Cytometry, Staining

    Activation of ATP release in frozen/thawed and rejuvenated RBCs from controls and OHSt patients. Suspensions of frozen/thawed and rejuvenated RBCs were exposed to isoosmotic (Iso) or hypoosmotic (Hypo, 240 mOsm/kg) stimulation. Experiments were performed with or without preincubation with 100 μM CBX, a PANX1 inhibitor. Results are expressed as eATP (pmol/10 6 cells). ( A ) Stacked graph showing total eATP including lytic eATP (hatched bars) released from control (white bars) or OHSt (grey bars) RBCs. The hemoglobin concentration of paired samples was used to estimate lytic eATP. ( B ) Non-lytic eATP was calculated as the differences between total eATP and lytic eATP. Total, lytic, and non-lytic eATP values were compared using a One-Way ANOVA Dunnett’s test for Multiple Comparison (N = 3). The results shown are mean values ± SEM. ( p values > 0.05: ns, ≤0.05: *, ≤0.01: **). ns stands for not significant.
    Figure Legend Snippet: Activation of ATP release in frozen/thawed and rejuvenated RBCs from controls and OHSt patients. Suspensions of frozen/thawed and rejuvenated RBCs were exposed to isoosmotic (Iso) or hypoosmotic (Hypo, 240 mOsm/kg) stimulation. Experiments were performed with or without preincubation with 100 μM CBX, a PANX1 inhibitor. Results are expressed as eATP (pmol/10 6 cells). ( A ) Stacked graph showing total eATP including lytic eATP (hatched bars) released from control (white bars) or OHSt (grey bars) RBCs. The hemoglobin concentration of paired samples was used to estimate lytic eATP. ( B ) Non-lytic eATP was calculated as the differences between total eATP and lytic eATP. Total, lytic, and non-lytic eATP values were compared using a One-Way ANOVA Dunnett’s test for Multiple Comparison (N = 3). The results shown are mean values ± SEM. ( p values > 0.05: ns, ≤0.05: *, ≤0.01: **). ns stands for not significant.

    Techniques Used: Activation Assay, Concentration Assay

    5(6)-carboxyfluorescein uptake in control (Ctrl) and OHSt RBCs after stimulation by high K + . ( A ) MFI due to the dye uptake in Ctrl (black) and OHSt (grey) frozen/thawed rejuvenated RBCs measured by flow cytometry in basal conditions and after stimulation by KCl without or with incubation with CBX. ( B ) Carbenoxolone (CBX), probenecid (PBC), and mefloquine (MFQ), three different PANX1 inhibitors, were added to RBC 20 min before the stimulation, resulting in a fluorescence signal similar to that of basal conditions. ( C ) FSC was also determined in all the conditions described above under KCl stimulation. Data obtained from stimulated cells were compared to that of unstimulated cells or cells stimulated in the presence of CBX using a paired Two-way Anova Sidak’s multiple comparisons test. The same test but unpaired was used when Ctrl and OHSt RBCs were compared. ( p values > 0.05: ns, ≤0.05: *, ≤0.0001: ****). ns stands for not significant.
    Figure Legend Snippet: 5(6)-carboxyfluorescein uptake in control (Ctrl) and OHSt RBCs after stimulation by high K + . ( A ) MFI due to the dye uptake in Ctrl (black) and OHSt (grey) frozen/thawed rejuvenated RBCs measured by flow cytometry in basal conditions and after stimulation by KCl without or with incubation with CBX. ( B ) Carbenoxolone (CBX), probenecid (PBC), and mefloquine (MFQ), three different PANX1 inhibitors, were added to RBC 20 min before the stimulation, resulting in a fluorescence signal similar to that of basal conditions. ( C ) FSC was also determined in all the conditions described above under KCl stimulation. Data obtained from stimulated cells were compared to that of unstimulated cells or cells stimulated in the presence of CBX using a paired Two-way Anova Sidak’s multiple comparisons test. The same test but unpaired was used when Ctrl and OHSt RBCs were compared. ( p values > 0.05: ns, ≤0.05: *, ≤0.0001: ****). ns stands for not significant.

    Techniques Used: Flow Cytometry, Incubation, Fluorescence

    Activation of ATP release in fresh RBCs by a hypoosmotic challenge. ( A ) RBC suspensions were exposed to isoosmotic (Iso) or hypoosmotic (Hypo, 240 mOsm/kg) stimulation. Experiments were performed with or without preincubation with 100 μM CBX, a PANX1 inhibitor. Results are expressed as eATP (pmol/10 6 cells). The hemoglobin concentration of paired samples was used to estimate lytic eATP. A stacked graph was built to show the contribution of lytic ATP (hatched bars) within total ATP release from RBCs. Lytic and total eATP values were compared using a One-Way ANOVA Dunnett’s test for Multiple Comparisons (N = 5). The results shown are mean values ± SEM. ( B ) The difference between total eATP and lytic eATP was calculated and denoted as non-lytic eATP. Non-lytic eATP values were compared using a One-Way ANOVA Dunnett’s test for Multiple Comparisons. ( C ) PS exposure was measured by flow cytometry on Lactadherin-FITC stained RBCs in basal conditions after treatment with Fenton’s reagent, as a control of eryptosis, and in basal or hypoosmotic conditions without Fenton. ( D ) Caspase activation was measured under conditions similar to PS exposure but on FLICA 660 caspase3/7-stained RBCs. The statistical analysis of the last two measurements (N = 5) was performed using an RM-one-way ANOVA test. ( p values > 0.05: ns, ≤0.05: *, ≤0.01: ** and ≤0.001: ***). ns stands for not significant.
    Figure Legend Snippet: Activation of ATP release in fresh RBCs by a hypoosmotic challenge. ( A ) RBC suspensions were exposed to isoosmotic (Iso) or hypoosmotic (Hypo, 240 mOsm/kg) stimulation. Experiments were performed with or without preincubation with 100 μM CBX, a PANX1 inhibitor. Results are expressed as eATP (pmol/10 6 cells). The hemoglobin concentration of paired samples was used to estimate lytic eATP. A stacked graph was built to show the contribution of lytic ATP (hatched bars) within total ATP release from RBCs. Lytic and total eATP values were compared using a One-Way ANOVA Dunnett’s test for Multiple Comparisons (N = 5). The results shown are mean values ± SEM. ( B ) The difference between total eATP and lytic eATP was calculated and denoted as non-lytic eATP. Non-lytic eATP values were compared using a One-Way ANOVA Dunnett’s test for Multiple Comparisons. ( C ) PS exposure was measured by flow cytometry on Lactadherin-FITC stained RBCs in basal conditions after treatment with Fenton’s reagent, as a control of eryptosis, and in basal or hypoosmotic conditions without Fenton. ( D ) Caspase activation was measured under conditions similar to PS exposure but on FLICA 660 caspase3/7-stained RBCs. The statistical analysis of the last two measurements (N = 5) was performed using an RM-one-way ANOVA test. ( p values > 0.05: ns, ≤0.05: *, ≤0.01: ** and ≤0.001: ***). ns stands for not significant.

    Techniques Used: Activation Assay, Concentration Assay, Flow Cytometry, Staining

    Stomatin and PANX1 proximity in control and OHSt RBCs assessed by Proximity Ligation Assay (PLA) and analyzed by Imaging Flow Cytometry. ( A ) PLA-positive (PLA+) events characterized by red spots which result from the detection oligos coupled to fluorochromes (FarRed) hybridizing to sequences in the amplified DNA were visualized by Imaging Flow Cytometry and merged with Brightfield images of the corresponding Control RBCs. ( B ) Quantification of the assay corresponding to the percentage of PLA+ RBCs was performed several times in different conditions: without any primary antibody (none, N = 10), with only one antibody (anti-stomatin or anti-PANX1 or anti-AE1, N = 3 for each condition) and in the presence of two antibodies (anti-stomatin + anti-PANX1 or anti-stomatin + anti-AE1, N = 7 in both conditions). A comparison was performed using an unpaired One-way Anova Tukey’s test. ( C ) PLA using anti-stomatin + anti-PANX1 was performed in identical conditions to those described above but on frozen/thawed control (Ctrl) and OHSt RBCs and compared using paired or unpaired (between control and OHSt) Two-way Anova Sidak’s multiple comparisons test ( p values > 0.05: ns, ≤0.05: *, ≤0.01: ** and ≤0.001: ***). ns stands for not significant.
    Figure Legend Snippet: Stomatin and PANX1 proximity in control and OHSt RBCs assessed by Proximity Ligation Assay (PLA) and analyzed by Imaging Flow Cytometry. ( A ) PLA-positive (PLA+) events characterized by red spots which result from the detection oligos coupled to fluorochromes (FarRed) hybridizing to sequences in the amplified DNA were visualized by Imaging Flow Cytometry and merged with Brightfield images of the corresponding Control RBCs. ( B ) Quantification of the assay corresponding to the percentage of PLA+ RBCs was performed several times in different conditions: without any primary antibody (none, N = 10), with only one antibody (anti-stomatin or anti-PANX1 or anti-AE1, N = 3 for each condition) and in the presence of two antibodies (anti-stomatin + anti-PANX1 or anti-stomatin + anti-AE1, N = 7 in both conditions). A comparison was performed using an unpaired One-way Anova Tukey’s test. ( C ) PLA using anti-stomatin + anti-PANX1 was performed in identical conditions to those described above but on frozen/thawed control (Ctrl) and OHSt RBCs and compared using paired or unpaired (between control and OHSt) Two-way Anova Sidak’s multiple comparisons test ( p values > 0.05: ns, ≤0.05: *, ≤0.01: ** and ≤0.001: ***). ns stands for not significant.

    Techniques Used: Proximity Ligation Assay, Imaging, Flow Cytometry, Amplification

    Stomatin and PANX1 expression in OHSt RBCs compared to controls. ( A ) Western blot analysis of RBC ghosts from 3 controls (C1, C2, C2) and the 3 OHSt patients (O1, O2, O3) using mouse monoclonal anti-stomatin (E-5), rabbit polyclonal anti-PANX1 and rabbit polyclonal anti-p55 as control. The molecular weights (kDa) of the detected bands are mentioned. ( B ) Flow cytometry analysis of stomatin expression in the RBCs of the 3 controls and the 3 OHSt patients after permeabilisation. Negative controls consist of the observed fluorescence with only the secondary antibody. The averages of Mean of Fluorescence Intensity (MFI) were compared between OHSt and control (Ctrl) RBCs using a two-way ANOVA Sidak’s multicomparison test ( p values > 0.05: ns and ≤0.05: *, ns stands for not significant), paired for comparisons within OHSt samples when signals with anti-stomatin were compared to that of Negative controls and unpaired when OHSt were compared to Ctrl. ( C ) Flow cytometry analysis of PANX1 expression in the 3 controls and the 3 OHSt patient RBCs after permeabilization and comparisons of MFIs between controls and OHSt, as performed in B.
    Figure Legend Snippet: Stomatin and PANX1 expression in OHSt RBCs compared to controls. ( A ) Western blot analysis of RBC ghosts from 3 controls (C1, C2, C2) and the 3 OHSt patients (O1, O2, O3) using mouse monoclonal anti-stomatin (E-5), rabbit polyclonal anti-PANX1 and rabbit polyclonal anti-p55 as control. The molecular weights (kDa) of the detected bands are mentioned. ( B ) Flow cytometry analysis of stomatin expression in the RBCs of the 3 controls and the 3 OHSt patients after permeabilisation. Negative controls consist of the observed fluorescence with only the secondary antibody. The averages of Mean of Fluorescence Intensity (MFI) were compared between OHSt and control (Ctrl) RBCs using a two-way ANOVA Sidak’s multicomparison test ( p values > 0.05: ns and ≤0.05: *, ns stands for not significant), paired for comparisons within OHSt samples when signals with anti-stomatin were compared to that of Negative controls and unpaired when OHSt were compared to Ctrl. ( C ) Flow cytometry analysis of PANX1 expression in the 3 controls and the 3 OHSt patient RBCs after permeabilization and comparisons of MFIs between controls and OHSt, as performed in B.

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

    4) Product Images from "Age Dependent Changes in Corneal Epithelial Cell Signaling"

    Article Title: Age Dependent Changes in Corneal Epithelial Cell Signaling

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2022.886721

    Inhibition of pannexin-1, P2X7, or P2Y2 have different effects on cellular trajectory at the wound edge but all decrease percent wound closure. Confluent Human Corneal Limbal Epithelial cell cultures were pre-incubated in the presence or absence of inhibitors, stained with the SiR Actin, scratch-wounded, and imaged at a rate of one frame per 5 min for 2 h using the Zeiss LSM 880 confocal microscope. (A) Inhibition of pannexin-1, P2X7, or P2Y2 led to significantly diminished wound closure at the 2 h time point. Two-tailed Student’s t-tests were performed to compare the percent wound closure of each inhibitor to an uninhibited control (* p
    Figure Legend Snippet: Inhibition of pannexin-1, P2X7, or P2Y2 have different effects on cellular trajectory at the wound edge but all decrease percent wound closure. Confluent Human Corneal Limbal Epithelial cell cultures were pre-incubated in the presence or absence of inhibitors, stained with the SiR Actin, scratch-wounded, and imaged at a rate of one frame per 5 min for 2 h using the Zeiss LSM 880 confocal microscope. (A) Inhibition of pannexin-1, P2X7, or P2Y2 led to significantly diminished wound closure at the 2 h time point. Two-tailed Student’s t-tests were performed to compare the percent wound closure of each inhibitor to an uninhibited control (* p

    Techniques Used: Inhibition, Incubation, Staining, Microscopy, Two Tailed Test

    Localization of pannexin-1 is elevated in corneas from younger mice at the wound edge. Enucleated globes were scratch wounded and allowed to heal for two or 5 h. Globes were fixed in 4% paraformaldehyde, corneas were dissected, and immunohistochemistry was performed to visualize localization of pannexin-1 near the wound edge (green). Imaging was performed using the Zeiss LSM 880 confocal microscope. Topographical maps denoting the intensity of staining throughout the region were generated from the imaging data. (A) In younger mice, pannexin-1 localization is diffuse at 2 h, with similar expression adjacent to and further away from the wound. This is seen in the topographical map as a relatively flat, green diagram. (B) In younger mice at 5 h, the increase in pannexin-1 near the wound edge is detected as red peaks in the topographical map. (C,D) Older mice have a similar localization of pannexin-1 throughout the epithelium at both two and 5 h. The data represent three eyes per condition, with each eye from one mouse. Scale bar is 50 microns.
    Figure Legend Snippet: Localization of pannexin-1 is elevated in corneas from younger mice at the wound edge. Enucleated globes were scratch wounded and allowed to heal for two or 5 h. Globes were fixed in 4% paraformaldehyde, corneas were dissected, and immunohistochemistry was performed to visualize localization of pannexin-1 near the wound edge (green). Imaging was performed using the Zeiss LSM 880 confocal microscope. Topographical maps denoting the intensity of staining throughout the region were generated from the imaging data. (A) In younger mice, pannexin-1 localization is diffuse at 2 h, with similar expression adjacent to and further away from the wound. This is seen in the topographical map as a relatively flat, green diagram. (B) In younger mice at 5 h, the increase in pannexin-1 near the wound edge is detected as red peaks in the topographical map. (C,D) Older mice have a similar localization of pannexin-1 throughout the epithelium at both two and 5 h. The data represent three eyes per condition, with each eye from one mouse. Scale bar is 50 microns.

    Techniques Used: Mouse Assay, Immunohistochemistry, Imaging, Microscopy, Staining, Generated, Expressing

    5) Product Images from "Age Dependent Changes in Corneal Epithelial Cell Signaling"

    Article Title: Age Dependent Changes in Corneal Epithelial Cell Signaling

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2022.886721

    Inhibition of pannexin-1, P2X7, or P2Y2 have different effects on cellular trajectory at the wound edge but all decrease percent wound closure. Confluent Human Corneal Limbal Epithelial cell cultures were pre-incubated in the presence or absence of inhibitors, stained with the SiR Actin, scratch-wounded, and imaged at a rate of one frame per 5 min for 2 h using the Zeiss LSM 880 confocal microscope. (A) Inhibition of pannexin-1, P2X7, or P2Y2 led to significantly diminished wound closure at the 2 h time point. Two-tailed Student’s t-tests were performed to compare the percent wound closure of each inhibitor to an uninhibited control (* p
    Figure Legend Snippet: Inhibition of pannexin-1, P2X7, or P2Y2 have different effects on cellular trajectory at the wound edge but all decrease percent wound closure. Confluent Human Corneal Limbal Epithelial cell cultures were pre-incubated in the presence or absence of inhibitors, stained with the SiR Actin, scratch-wounded, and imaged at a rate of one frame per 5 min for 2 h using the Zeiss LSM 880 confocal microscope. (A) Inhibition of pannexin-1, P2X7, or P2Y2 led to significantly diminished wound closure at the 2 h time point. Two-tailed Student’s t-tests were performed to compare the percent wound closure of each inhibitor to an uninhibited control (* p

    Techniques Used: Inhibition, Incubation, Staining, Microscopy, Two Tailed Test

    Localization of pannexin-1 is elevated in corneas from younger mice at the wound edge. Enucleated globes were scratch wounded and allowed to heal for two or 5 h. Globes were fixed in 4% paraformaldehyde, corneas were dissected, and immunohistochemistry was performed to visualize localization of pannexin-1 near the wound edge (green). Imaging was performed using the Zeiss LSM 880 confocal microscope. Topographical maps denoting the intensity of staining throughout the region were generated from the imaging data. (A) In younger mice, pannexin-1 localization is diffuse at 2 h, with similar expression adjacent to and further away from the wound. This is seen in the topographical map as a relatively flat, green diagram. (B) In younger mice at 5 h, the increase in pannexin-1 near the wound edge is detected as red peaks in the topographical map. (C,D) Older mice have a similar localization of pannexin-1 throughout the epithelium at both two and 5 h. The data represent three eyes per condition, with each eye from one mouse. Scale bar is 50 microns.
    Figure Legend Snippet: Localization of pannexin-1 is elevated in corneas from younger mice at the wound edge. Enucleated globes were scratch wounded and allowed to heal for two or 5 h. Globes were fixed in 4% paraformaldehyde, corneas were dissected, and immunohistochemistry was performed to visualize localization of pannexin-1 near the wound edge (green). Imaging was performed using the Zeiss LSM 880 confocal microscope. Topographical maps denoting the intensity of staining throughout the region were generated from the imaging data. (A) In younger mice, pannexin-1 localization is diffuse at 2 h, with similar expression adjacent to and further away from the wound. This is seen in the topographical map as a relatively flat, green diagram. (B) In younger mice at 5 h, the increase in pannexin-1 near the wound edge is detected as red peaks in the topographical map. (C,D) Older mice have a similar localization of pannexin-1 throughout the epithelium at both two and 5 h. The data represent three eyes per condition, with each eye from one mouse. Scale bar is 50 microns.

    Techniques Used: Mouse Assay, Immunohistochemistry, Imaging, Microscopy, Staining, Generated, Expressing

    6) Product Images from "Synaptic Scaffolds, Ion Channels and Polyamines in Mouse Photoreceptor Synapses: Anatomy of a Signaling Complex"

    Article Title: Synaptic Scaffolds, Ion Channels and Polyamines in Mouse Photoreceptor Synapses: Anatomy of a Signaling Complex

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2021.667046

    Pannexin labeling in mouse retina. (A) Immunostaining for Pannexin 1 in mouse retina revealed strong labeling in the outer plexiform layer and weaker labeling in the inner nuclear layer and ganglion cell layer. Three μm confocal stack. Labels for retinal nuclear layers are as in Figure 1 . (B) Immunostaining for Pannexin 2 displayed strong labeling in the OPL and weaker labeling in the INL and photoreceptor layer. Three μm confocal stack. (C) Average mRNA expression levels of Pannexin1 and Pannexin2 in mouse retina from single-cell transcriptome data. In the OPL, the most prominent expression of Pannexin1 is in Off cone bipolar cells, with very weak expression in horizontal cells. Pannexin2 expression is largely absent from horizontal cells, but weakly present in On and Off bipolar cells. Data adapted from Hoang et al. (2020) .
    Figure Legend Snippet: Pannexin labeling in mouse retina. (A) Immunostaining for Pannexin 1 in mouse retina revealed strong labeling in the outer plexiform layer and weaker labeling in the inner nuclear layer and ganglion cell layer. Three μm confocal stack. Labels for retinal nuclear layers are as in Figure 1 . (B) Immunostaining for Pannexin 2 displayed strong labeling in the OPL and weaker labeling in the INL and photoreceptor layer. Three μm confocal stack. (C) Average mRNA expression levels of Pannexin1 and Pannexin2 in mouse retina from single-cell transcriptome data. In the OPL, the most prominent expression of Pannexin1 is in Off cone bipolar cells, with very weak expression in horizontal cells. Pannexin2 expression is largely absent from horizontal cells, but weakly present in On and Off bipolar cells. Data adapted from Hoang et al. (2020) .

    Techniques Used: Labeling, Immunostaining, Expressing

    7) Product Images from "Pannexin1: Role as a Sensor to Injury Is Attenuated in Pretype 2 Corneal Diabetic Epithelium"

    Article Title: Pannexin1: Role as a Sensor to Injury Is Attenuated in Pretype 2 Corneal Diabetic Epithelium

    Journal: Analytical Cellular Pathology (Amsterdam)

    doi: 10.1155/2021/4793338

    Pannexin1 mediates communication between corneal epithelial cells. (a) Representative images of B6 control corneas incubated in the presence of 10Panx or a scrambled control (control) peptide and then labelled with Cal-520 AM and CellMask Deep Red for imaging. Representative images of the Ca 2+ wave in basal epithelial cells after injury: 50, 100, and 150 seconds. Arrowheads mark cells to follow over time. (b) MATLAB event charts showing the Ca 2+ mobilization events detected over time. (c) Event probability values comparing corneas preincubated in the presence of either 10Panx or scrambled control (control) peptide followed by an epithelial scratch injury. (d) Event probability values comparing the wound response near the leading edge of the corneal epithelium ex vivo to in vitro following a scratch wound. Data are expressed as mean ± SEM and were analyzed with a one-way ANOVA with the Tukey's multiple comparison test. p
    Figure Legend Snippet: Pannexin1 mediates communication between corneal epithelial cells. (a) Representative images of B6 control corneas incubated in the presence of 10Panx or a scrambled control (control) peptide and then labelled with Cal-520 AM and CellMask Deep Red for imaging. Representative images of the Ca 2+ wave in basal epithelial cells after injury: 50, 100, and 150 seconds. Arrowheads mark cells to follow over time. (b) MATLAB event charts showing the Ca 2+ mobilization events detected over time. (c) Event probability values comparing corneas preincubated in the presence of either 10Panx or scrambled control (control) peptide followed by an epithelial scratch injury. (d) Event probability values comparing the wound response near the leading edge of the corneal epithelium ex vivo to in vitro following a scratch wound. Data are expressed as mean ± SEM and were analyzed with a one-way ANOVA with the Tukey's multiple comparison test. p

    Techniques Used: Incubation, Imaging, Ex Vivo, In Vitro

    Association of pannexin1 and P2X7 in unwounded corneas and 2 hrs after injury in DiO and B6 control corneal epithelium. Proximity ligation assays were performed using antibodies to P2X7 and pannexin1. (a) Representative enface and orthogonal (ortho) images of B6 control and DiO corneas are shown. The green color displays puncta of association. Corneal epithelium was counter stained with rhodamine phalloidin (asterisk marks wound area). The numbers and letters mark the individual cells that were analyzed using CellProfiler near the wound and back from the wound. The boxes indicate the representative individual cells that are shown in (b) after PLA analysis with CellProfiler. Bar equals 20 microns (PLA enface and actin) or 15 microns (PLA ortho). (b) Analysis of overlapping puncta for each condition (unwounded, leading edge, back from leading edge) was performed and a box plot drawn using R. The mean puncta for each region are placed above the box. Mean ± SEM are plotted, and two-way ANOVA with Tukey's multiple comparison of means was performed. p
    Figure Legend Snippet: Association of pannexin1 and P2X7 in unwounded corneas and 2 hrs after injury in DiO and B6 control corneal epithelium. Proximity ligation assays were performed using antibodies to P2X7 and pannexin1. (a) Representative enface and orthogonal (ortho) images of B6 control and DiO corneas are shown. The green color displays puncta of association. Corneal epithelium was counter stained with rhodamine phalloidin (asterisk marks wound area). The numbers and letters mark the individual cells that were analyzed using CellProfiler near the wound and back from the wound. The boxes indicate the representative individual cells that are shown in (b) after PLA analysis with CellProfiler. Bar equals 20 microns (PLA enface and actin) or 15 microns (PLA ortho). (b) Analysis of overlapping puncta for each condition (unwounded, leading edge, back from leading edge) was performed and a box plot drawn using R. The mean puncta for each region are placed above the box. Mean ± SEM are plotted, and two-way ANOVA with Tukey's multiple comparison of means was performed. p

    Techniques Used: Ligation, Staining, Proximity Ligation Assay

    Pannexin1 localization in B6 control and DiO corneal epithelium. (a) Representative confocal immunofluorescent images of pannexin1 in unwounded and wounded corneas: 2, 8, and 20 hrs postepithelial debridement. Dotted line indicates the leading edge. Bar equals 25 microns. (b) Enlarged immunofluorescent images of the rectangular boxes in (a) (20 hrs), which show the cell morphology back from the leading edge. Asterisks ( ∗ ) mark representative cells in both B6 control and DiO ~6 cells back from the leading edge. Note the elongation of the ∗ cell in DiO. Bar equals 10 microns. N is a minimum of 3 independent experiments.
    Figure Legend Snippet: Pannexin1 localization in B6 control and DiO corneal epithelium. (a) Representative confocal immunofluorescent images of pannexin1 in unwounded and wounded corneas: 2, 8, and 20 hrs postepithelial debridement. Dotted line indicates the leading edge. Bar equals 25 microns. (b) Enlarged immunofluorescent images of the rectangular boxes in (a) (20 hrs), which show the cell morphology back from the leading edge. Asterisks ( ∗ ) mark representative cells in both B6 control and DiO ~6 cells back from the leading edge. Note the elongation of the ∗ cell in DiO. Bar equals 10 microns. N is a minimum of 3 independent experiments.

    Techniques Used:

    Pannexin1 mediates corneal epithelial sheet migration. Representative images of B6 control corneas (in the presence of 10Panx or a scrambled control (control) peptide) wounded, incubated for 2 hrs in medium, fixed, and then stained with rhodamine phalloidin. The inset highlights the differences in actin at the leading edge and demonstrates coalescence of actin in the 10Panx treated corneas. Bar equals 30 microns. N is a minimum of 3 independent experiments.
    Figure Legend Snippet: Pannexin1 mediates corneal epithelial sheet migration. Representative images of B6 control corneas (in the presence of 10Panx or a scrambled control (control) peptide) wounded, incubated for 2 hrs in medium, fixed, and then stained with rhodamine phalloidin. The inset highlights the differences in actin at the leading edge and demonstrates coalescence of actin in the 10Panx treated corneas. Bar equals 30 microns. N is a minimum of 3 independent experiments.

    Techniques Used: Migration, Incubation, Staining

    8) Product Images from "Focused Ultrasound Stimulates ER Localized Mechanosensitive PANNEXIN-1 to Mediate Intracellular Calcium Release in Invasive Cancer Cells"

    Article Title: Focused Ultrasound Stimulates ER Localized Mechanosensitive PANNEXIN-1 to Mediate Intracellular Calcium Release in Invasive Cancer Cells

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2020.00504

    Treatment of PC-3 cells with 10 PX1 (PANX1 inhibitor) abolishes the normal FUS-induced Ca 2 + oscillation response but uncovers single Ca 2 + transients. (A) Left column, the cells exhibited strong Ca 2+ responses at 20 min after 200 μM scrambled peptide application as a control. Center column, cells were stimulated at 20 min after 200 μM 10 Panx1 peptide ( 10 PX1) application, and the responses were partly reduced. Right column, 20 min after 2 μM Xestospongin C (XC) application, the responses were also partly reduced. Two representative cells were shown in each treatment. (B) Quantitative CRI values of the inhibitor treatments. n = 3 (XC), or n = 6 (SC, 10 PX1). Error bars, s.e.m., ANOVA, Dunnet’s correction, exact p -values. (C) Fluorescence patterns in cells that first responded to the stimulus after the treatments. Two representative cells are shown by ΔF/F. (D) Fluorescence patterns in several cells that first responded to the stimulus after the treatments; Scrambled (9 cells), 10 PX1 (5 cells) and XC (6 cells).
    Figure Legend Snippet: Treatment of PC-3 cells with 10 PX1 (PANX1 inhibitor) abolishes the normal FUS-induced Ca 2 + oscillation response but uncovers single Ca 2 + transients. (A) Left column, the cells exhibited strong Ca 2+ responses at 20 min after 200 μM scrambled peptide application as a control. Center column, cells were stimulated at 20 min after 200 μM 10 Panx1 peptide ( 10 PX1) application, and the responses were partly reduced. Right column, 20 min after 2 μM Xestospongin C (XC) application, the responses were also partly reduced. Two representative cells were shown in each treatment. (B) Quantitative CRI values of the inhibitor treatments. n = 3 (XC), or n = 6 (SC, 10 PX1). Error bars, s.e.m., ANOVA, Dunnet’s correction, exact p -values. (C) Fluorescence patterns in cells that first responded to the stimulus after the treatments. Two representative cells are shown by ΔF/F. (D) Fluorescence patterns in several cells that first responded to the stimulus after the treatments; Scrambled (9 cells), 10 PX1 (5 cells) and XC (6 cells).

    Techniques Used: Fluorescence

    (A) Schematic of new working model FUS-dependent response mechanisms in PC-3 invasive cancer cells. (1) FUS stimulation activates ER localized mechanosensitive PANX1 resulting in internal Ca 2+ release from ER stores. (2) This cytoplasmic Ca 2+ signal stimulates ATP release through PANX1 PM channels. (3) The released ATP acts on purineregic receptors, many in adjacent cells. (4) This results in a propagating extracellular Ca 2+ wave which spreads through the cell population possibly via PM PANX1 or opening of PM Ca 2+ channels. (5) FUS stimulation also results in secretion of cytokines/chemokines. (B) Schematic of currently accepted working model based largely on conventional US stimulation with no proposed role for internal ER Ca 2+ release but rather a link to US energy transduction to ER mediated by IP 3 R. MB, Microbubbles.
    Figure Legend Snippet: (A) Schematic of new working model FUS-dependent response mechanisms in PC-3 invasive cancer cells. (1) FUS stimulation activates ER localized mechanosensitive PANX1 resulting in internal Ca 2+ release from ER stores. (2) This cytoplasmic Ca 2+ signal stimulates ATP release through PANX1 PM channels. (3) The released ATP acts on purineregic receptors, many in adjacent cells. (4) This results in a propagating extracellular Ca 2+ wave which spreads through the cell population possibly via PM PANX1 or opening of PM Ca 2+ channels. (5) FUS stimulation also results in secretion of cytokines/chemokines. (B) Schematic of currently accepted working model based largely on conventional US stimulation with no proposed role for internal ER Ca 2+ release but rather a link to US energy transduction to ER mediated by IP 3 R. MB, Microbubbles.

    Techniques Used: Transduction

    Localization of PANX1. (A,B) Schematic of fluorescent WT and mt PANX1 constructs (A) and the N and C-terminal epitopes recognized by anti-PANX1 antibodies (Ab) (B) . (C) Localization of WT PANX1-EGFP and mt PANX1-mRFP in transfected HEK cells. (D) Localization of endogenous PANX1 in PC-3 cells using N- or C-terminal specific Abs. Nuclear DAPI stain is depicted as blue. (E) TIRF imaging on HEK cells transfected by WT PANX1-EGFP or mt PANX1-mRFP constructs. WT PANX1-EGFP localizes in the PM and the ER, while mt PANX1-mRFP only in the ER.
    Figure Legend Snippet: Localization of PANX1. (A,B) Schematic of fluorescent WT and mt PANX1 constructs (A) and the N and C-terminal epitopes recognized by anti-PANX1 antibodies (Ab) (B) . (C) Localization of WT PANX1-EGFP and mt PANX1-mRFP in transfected HEK cells. (D) Localization of endogenous PANX1 in PC-3 cells using N- or C-terminal specific Abs. Nuclear DAPI stain is depicted as blue. (E) TIRF imaging on HEK cells transfected by WT PANX1-EGFP or mt PANX1-mRFP constructs. WT PANX1-EGFP localizes in the PM and the ER, while mt PANX1-mRFP only in the ER.

    Techniques Used: Construct, Transfection, Staining, Imaging

    PANX1 expression appears to be both necessary and sufficient for intracellular Ca 2 + responses. (A) si-PANX1 RNA treatment in PC3 cells reduced Ca 2+ responses compared to si-negative RNA (scramble) as a control. (B) Quantitative cell response index (CRI) values of the si-PANX1 RNA treatments relative to the control. n = 3. Error bars, s.e.m., exact p -values by a two-tailed t -test. (C) Fluorescence patterns in cells that first responded to the FUS stimulus after the treatments. One representative cell (top) is shown with fluorescence patterns in ten cells (bottom). (D) HEK293T cells transfected with WT PANX1 or mt PANX1 1–89 -mRFP (mt PANX1-mRFP) constructs showed Ca 2+ responses while control HEK cells transfected with dsRED construct have no FUS-induced Ca 2+ response. (E) Quantitative CRI values of the transfected cells. n = 3. Error bars, s.e.m., ANOVA, Dunnet’s correction, exact p values.
    Figure Legend Snippet: PANX1 expression appears to be both necessary and sufficient for intracellular Ca 2 + responses. (A) si-PANX1 RNA treatment in PC3 cells reduced Ca 2+ responses compared to si-negative RNA (scramble) as a control. (B) Quantitative cell response index (CRI) values of the si-PANX1 RNA treatments relative to the control. n = 3. Error bars, s.e.m., exact p -values by a two-tailed t -test. (C) Fluorescence patterns in cells that first responded to the FUS stimulus after the treatments. One representative cell (top) is shown with fluorescence patterns in ten cells (bottom). (D) HEK293T cells transfected with WT PANX1 or mt PANX1 1–89 -mRFP (mt PANX1-mRFP) constructs showed Ca 2+ responses while control HEK cells transfected with dsRED construct have no FUS-induced Ca 2+ response. (E) Quantitative CRI values of the transfected cells. n = 3. Error bars, s.e.m., ANOVA, Dunnet’s correction, exact p values.

    Techniques Used: Expressing, Two Tailed Test, Fluorescence, Transfection, Construct

    Effects of intercellular Ca 2 + wave inhibitors on Ca 2 + response (A–D) , and effects of PANX1 modulation on ATP release (E–G) . The responses are represented when PC-3 cells were treated with Apyrase, Suramin, PPADS and mock; Mock (A) , 20–50 units/ml Apyrase (B) , 100 μM Suramin (C) , and 100 μM PPADS (D) . The percentage of responding cells over time was shown. Experimental results presented are representative and were independently replicates at least two times with three independent biological samples. (E) Quantification of PANX1-mediated ATP release from PC-3 cells pretreated for 15 min with scrambled (SC), 10 PX1, or Xestospongin C (XC). n = 4. ANOVA, Dunnet’s correction, exact p -values. (F) Quantification of PANX1-mediated ATP release from PC-3 cells transfected with control si-negative or si-PANX1 RNA. n = 4, p -values by a two-tailed t -test. (G) Quantification of PANX1-mediated ATP release from HEK cells transfected with dsRED, mt PANX1-mRFP or WT PANX1-EGFP, and pretreated for 10 min with CBX (500 μM). n = 3. ANOVA, Dunnet’s correction, exact p -values. Error bars, s.e.m., NS, not significant.
    Figure Legend Snippet: Effects of intercellular Ca 2 + wave inhibitors on Ca 2 + response (A–D) , and effects of PANX1 modulation on ATP release (E–G) . The responses are represented when PC-3 cells were treated with Apyrase, Suramin, PPADS and mock; Mock (A) , 20–50 units/ml Apyrase (B) , 100 μM Suramin (C) , and 100 μM PPADS (D) . The percentage of responding cells over time was shown. Experimental results presented are representative and were independently replicates at least two times with three independent biological samples. (E) Quantification of PANX1-mediated ATP release from PC-3 cells pretreated for 15 min with scrambled (SC), 10 PX1, or Xestospongin C (XC). n = 4. ANOVA, Dunnet’s correction, exact p -values. (F) Quantification of PANX1-mediated ATP release from PC-3 cells transfected with control si-negative or si-PANX1 RNA. n = 4, p -values by a two-tailed t -test. (G) Quantification of PANX1-mediated ATP release from HEK cells transfected with dsRED, mt PANX1-mRFP or WT PANX1-EGFP, and pretreated for 10 min with CBX (500 μM). n = 3. ANOVA, Dunnet’s correction, exact p -values. Error bars, s.e.m., NS, not significant.

    Techniques Used: Transfection, Two Tailed Test

    9) Product Images from "Focused ultrasound stimulates ER localized mechanosensitive PANNEXIN-1 to mediate intracellular calcium release in invasive cancer cells"

    Article Title: Focused ultrasound stimulates ER localized mechanosensitive PANNEXIN-1 to mediate intracellular calcium release in invasive cancer cells

    Journal: bioRxiv

    doi: 10.1101/2020.04.03.024372

    (A). Schematic of new working model FUS-dependent response mechanisms in PC-3 invasive cancer cells. 1) FUS stimulation activates ER localized mechanosensitive PANX1 resulting in internal Ca 2+ release from ER stores. 2) This cytoplasmic Ca 2+ signal stimulates ATP release through PANX1 PM channels. 3) The released ATP acts on purineregic receptors, many in adjacent cells. 4) This results in a propagating extracellular Ca 2+ wave which spreads through the cell population possibly via PM PANX1 or opening of PM Ca 2+ channels. 5) FUS stimulation also results in secretion of cytokines/chemokines. (B) Schematic of currently accepted working model based largely on conventional US stimulation with no proposed role for internal ER Ca 2+ release but rather a link to US energy transduction to ER mediated by IP 3 R. MB, Microbubbles.
    Figure Legend Snippet: (A). Schematic of new working model FUS-dependent response mechanisms in PC-3 invasive cancer cells. 1) FUS stimulation activates ER localized mechanosensitive PANX1 resulting in internal Ca 2+ release from ER stores. 2) This cytoplasmic Ca 2+ signal stimulates ATP release through PANX1 PM channels. 3) The released ATP acts on purineregic receptors, many in adjacent cells. 4) This results in a propagating extracellular Ca 2+ wave which spreads through the cell population possibly via PM PANX1 or opening of PM Ca 2+ channels. 5) FUS stimulation also results in secretion of cytokines/chemokines. (B) Schematic of currently accepted working model based largely on conventional US stimulation with no proposed role for internal ER Ca 2+ release but rather a link to US energy transduction to ER mediated by IP 3 R. MB, Microbubbles.

    Techniques Used: Transduction

    Treatment of PC-3 cells with 10 PX1 (PANX1 inhibitor) abolishes the normal FUS-induced Ca 2+ oscillation response but uncovers single Ca 2+ transients. (A) Left column, the cells exhibited strong Ca 2+ responses at 20 min after 200 μM scrambled peptide application as a control. Center column, cells were stimulated at 20 min after 200 μM 10 Panx1 peptide ( 10 PX1) application, and the responses were partly reduced. Right column, 20 min after 2 μM Xestospongin C (XC) application, the responses were also partly reduced. Two representative cells were shown in each treatment. (B) Quantitative CRI values of the inhibitor treatments. n =3 (XC), or n =6 (SC, 10 PX1). Error bars, s.e.m., ANOVA, Dunnet’s correction, exact p values. (C) Fluorescence patterns in cells that first responded to the stimulus after the treatments. Two representative cells are shown by ΔF/F. (D) Fluorescence patterns in several cells that first responded to the stimulus after the treatments; Scrambled (9 cells), 10 PX1 (5 cells) and XC (6 cells).
    Figure Legend Snippet: Treatment of PC-3 cells with 10 PX1 (PANX1 inhibitor) abolishes the normal FUS-induced Ca 2+ oscillation response but uncovers single Ca 2+ transients. (A) Left column, the cells exhibited strong Ca 2+ responses at 20 min after 200 μM scrambled peptide application as a control. Center column, cells were stimulated at 20 min after 200 μM 10 Panx1 peptide ( 10 PX1) application, and the responses were partly reduced. Right column, 20 min after 2 μM Xestospongin C (XC) application, the responses were also partly reduced. Two representative cells were shown in each treatment. (B) Quantitative CRI values of the inhibitor treatments. n =3 (XC), or n =6 (SC, 10 PX1). Error bars, s.e.m., ANOVA, Dunnet’s correction, exact p values. (C) Fluorescence patterns in cells that first responded to the stimulus after the treatments. Two representative cells are shown by ΔF/F. (D) Fluorescence patterns in several cells that first responded to the stimulus after the treatments; Scrambled (9 cells), 10 PX1 (5 cells) and XC (6 cells).

    Techniques Used: Fluorescence

    Effects of intercellular Ca 2+ wave inhibitors on Ca 2+ response (A-D), and effects of PANX1 modulation on ATP release (E-G). The responses are represented when PC-3 cells were treated with Apyrase, Suramin, PPADS and mock; Mock (A) , 20-50 units/ml Apyrase (B) , 100 μM Suramin (C) , and 100 uM PPADS (D) . The percentage of responding cells over time was shown. Experimental results presented are representative and were independently replicates at least two times with three independent biological samples. (E) Quantification of PANX1-mediated ATP release from PC-3 cells pretreated for 15 min with scrambled (SC), 10 PX1, or Xestospongin C (XC). n =4. ANOVA, Dunnet’s correction, exact p values. (F) Quantification of PANX1-mediated ATP release from PC-3 cells transfected with control si-negative or si-PANX1 RNA. n =4, p values by a two-tailed t-test. (G) Quantification of PANX1-mediated ATP release from HEK cells transfected with dsRED, mt PANX1-mRFP or WT PANX1-EGFP, and pretreated for 10 min with CBX (500uM). n =3. ANOVA, Dunnet’s correction, exact p values. Error bars, s.e.m., NS, not significant.
    Figure Legend Snippet: Effects of intercellular Ca 2+ wave inhibitors on Ca 2+ response (A-D), and effects of PANX1 modulation on ATP release (E-G). The responses are represented when PC-3 cells were treated with Apyrase, Suramin, PPADS and mock; Mock (A) , 20-50 units/ml Apyrase (B) , 100 μM Suramin (C) , and 100 uM PPADS (D) . The percentage of responding cells over time was shown. Experimental results presented are representative and were independently replicates at least two times with three independent biological samples. (E) Quantification of PANX1-mediated ATP release from PC-3 cells pretreated for 15 min with scrambled (SC), 10 PX1, or Xestospongin C (XC). n =4. ANOVA, Dunnet’s correction, exact p values. (F) Quantification of PANX1-mediated ATP release from PC-3 cells transfected with control si-negative or si-PANX1 RNA. n =4, p values by a two-tailed t-test. (G) Quantification of PANX1-mediated ATP release from HEK cells transfected with dsRED, mt PANX1-mRFP or WT PANX1-EGFP, and pretreated for 10 min with CBX (500uM). n =3. ANOVA, Dunnet’s correction, exact p values. Error bars, s.e.m., NS, not significant.

    Techniques Used: Transfection, Two Tailed Test

    Localization of PANX1. (A, B) Schematic of fluorescent WT and mt PANX1 constructs ( A ) and the N and C-terminal epitopes recognized by anti-PANX1 antibodies (Ab) ( B ). (C) Localization of WT PANX1-EGFP and mt PANX1-mRFP in transfected HEK cells. (D) Localization of endogenous PANX1 in PC-3 cells using N- or C-terminal specific Abs. Nuclear DAPI stain is depicted as blue. (E) TIRF imaging on HEK cells transfected by WT PANX1-EGFP or mt PANX1-mRFP constructs. WT PANX1-EGFP localizes in the PM and the ER, while mt PANX1-mRFP only in the ER.
    Figure Legend Snippet: Localization of PANX1. (A, B) Schematic of fluorescent WT and mt PANX1 constructs ( A ) and the N and C-terminal epitopes recognized by anti-PANX1 antibodies (Ab) ( B ). (C) Localization of WT PANX1-EGFP and mt PANX1-mRFP in transfected HEK cells. (D) Localization of endogenous PANX1 in PC-3 cells using N- or C-terminal specific Abs. Nuclear DAPI stain is depicted as blue. (E) TIRF imaging on HEK cells transfected by WT PANX1-EGFP or mt PANX1-mRFP constructs. WT PANX1-EGFP localizes in the PM and the ER, while mt PANX1-mRFP only in the ER.

    Techniques Used: Construct, Transfection, Staining, Imaging

    PANX1 expression appears to be both necessary and sufficient for intracellular Ca 2+ responses. (A) si-PANX1 RNA treatment in PC3 cells reduced Ca 2+ responses compared to si-negative RNA (scramble) as a control. (B) Quantitative cell response index (CRI) values of the si-PANX1 RNA treatments relative to the control. n =3. Error bars, s.e.m., exact p values by a two-tailed t-test. (C) Fluorescence patterns in cells that first responded to the FUS stimulus after the treatments. One representative cell (top) is shown with fluorescence patterns in ten cells (bottom). (D) HEK293T cells transfected with WT PANX1 or mt PANX1 1-89 -mRFP (mt PANX1-mRFP) constructs showed Ca 2+ responses while control HEK cells transfected with dsRED construct have no FUS-induced Ca 2+ response. (E) Quantitative CRI values of the transfected cells. n =3. Error bars, s.e.m., ANOVA, Dunnet’s correction, exact p values.
    Figure Legend Snippet: PANX1 expression appears to be both necessary and sufficient for intracellular Ca 2+ responses. (A) si-PANX1 RNA treatment in PC3 cells reduced Ca 2+ responses compared to si-negative RNA (scramble) as a control. (B) Quantitative cell response index (CRI) values of the si-PANX1 RNA treatments relative to the control. n =3. Error bars, s.e.m., exact p values by a two-tailed t-test. (C) Fluorescence patterns in cells that first responded to the FUS stimulus after the treatments. One representative cell (top) is shown with fluorescence patterns in ten cells (bottom). (D) HEK293T cells transfected with WT PANX1 or mt PANX1 1-89 -mRFP (mt PANX1-mRFP) constructs showed Ca 2+ responses while control HEK cells transfected with dsRED construct have no FUS-induced Ca 2+ response. (E) Quantitative CRI values of the transfected cells. n =3. Error bars, s.e.m., ANOVA, Dunnet’s correction, exact p values.

    Techniques Used: Expressing, Two Tailed Test, Fluorescence, Transfection, Construct

    10) Product Images from "Pannexin1 links lymphatic function to lipid metabolism and atherosclerosis"

    Article Title: Pannexin1 links lymphatic function to lipid metabolism and atherosclerosis

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-14130-4

    Reduced atherosclerotic plaque stability in mice lacking Panx1. Representative images and quantification of (immuno-)stainings for CD68 ( A , E; brown signal), necrotic core ( B , F; yellow-marked acellular areas), collagen ( C , G; red signal) and α-SMA
    Figure Legend Snippet: Reduced atherosclerotic plaque stability in mice lacking Panx1. Representative images and quantification of (immuno-)stainings for CD68 ( A , E; brown signal), necrotic core ( B , F; yellow-marked acellular areas), collagen ( C , G; red signal) and α-SMA

    Techniques Used: Mouse Assay

    Panx1 deficiency reduces lymphatic function. ( A ) Panx1 expression in aortic ECs and LECs from WT mice was assessed by real-time qPCR (n = 3–4). ( B ) Percentage of resident and migratory DCs in CH draining lymph nodes of control
    Figure Legend Snippet: Panx1 deficiency reduces lymphatic function. ( A ) Panx1 expression in aortic ECs and LECs from WT mice was assessed by real-time qPCR (n = 3–4). ( B ) Percentage of resident and migratory DCs in CH draining lymph nodes of control

    Techniques Used: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction

    Targeted deletion of Panx1 in endothelial and monocytic cells favors atherosclerotic plaque development. Representative images of Panx1 immunofluorescent staining in ECs of a mouse carotid artery ( A and B ) and in macrophage foam cells within a carotid
    Figure Legend Snippet: Targeted deletion of Panx1 in endothelial and monocytic cells favors atherosclerotic plaque development. Representative images of Panx1 immunofluorescent staining in ECs of a mouse carotid artery ( A and B ) and in macrophage foam cells within a carotid

    Techniques Used: Staining

    Ubiquitous Panx1 deletion does not affect atherogenesis. Sudan-IV staining ( A ) and quantification of atherosclerotic lesion extent in the thoracic-abdominal aortas ( B ) and in the aortic roots ( C ) of Apoe −/− (white bars) and Panx1 −/−
    Figure Legend Snippet: Ubiquitous Panx1 deletion does not affect atherogenesis. Sudan-IV staining ( A ) and quantification of atherosclerotic lesion extent in the thoracic-abdominal aortas ( B ) and in the aortic roots ( C ) of Apoe −/− (white bars) and Panx1 −/−

    Techniques Used: Staining

    11) Product Images from "Pannexin1 links lymphatic function to lipid metabolism and atherosclerosis"

    Article Title: Pannexin1 links lymphatic function to lipid metabolism and atherosclerosis

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-14130-4

    Reduced atherosclerotic plaque stability in mice lacking Panx1. Representative images and quantification of (immuno-)stainings for CD68 ( A , E; brown signal), necrotic core ( B , F; yellow-marked acellular areas), collagen ( C , G; red signal) and α-SMA ( D , H; green signal) performed on aortic roots of Apoe −/− (white bars) and Panx1 −/− Apoe −/− (grey bars) mice after 5 weeks of HCD (n = 10). MCP-1-induced chemotaxis ( I ) and adhesion onto collagen-coated ( J ) or fibronectin-coated ( K ) surfaces of Panx1 fl/fl Apoe −/− (white bars) and Panx1 del Apoe −/− (black bars) BMDMs (n = 6). Number of apoptotic cells ( L ) using TUNEL staining or proliferating cells ( M ) using PCNA staining performed on aortic roots of Apoe −/− (white bars) and Panx1 −/− Apoe −/− (grey bars) mice after 5 weeks of HCD (n = 10). Scale bars represent 200 μm for A , or 100 μm for B , C , and D .
    Figure Legend Snippet: Reduced atherosclerotic plaque stability in mice lacking Panx1. Representative images and quantification of (immuno-)stainings for CD68 ( A , E; brown signal), necrotic core ( B , F; yellow-marked acellular areas), collagen ( C , G; red signal) and α-SMA ( D , H; green signal) performed on aortic roots of Apoe −/− (white bars) and Panx1 −/− Apoe −/− (grey bars) mice after 5 weeks of HCD (n = 10). MCP-1-induced chemotaxis ( I ) and adhesion onto collagen-coated ( J ) or fibronectin-coated ( K ) surfaces of Panx1 fl/fl Apoe −/− (white bars) and Panx1 del Apoe −/− (black bars) BMDMs (n = 6). Number of apoptotic cells ( L ) using TUNEL staining or proliferating cells ( M ) using PCNA staining performed on aortic roots of Apoe −/− (white bars) and Panx1 −/− Apoe −/− (grey bars) mice after 5 weeks of HCD (n = 10). Scale bars represent 200 μm for A , or 100 μm for B , C , and D .

    Techniques Used: Mouse Assay, Chemotaxis Assay, TUNEL Assay, Staining

    Panx1 deficiency reduces lymphatic function. ( A ) Panx1 expression in aortic ECs and LECs from WT mice was assessed by real-time qPCR (n = 3–4). ( B ) Percentage of resident and migratory DCs in CH draining lymph nodes of control (white bars) and Panx1 −/− mice (grey bars) (n = 5). ( C ) Representative images of lymphatic drainage 1 and 15 min after injection of 5 μl of Evans Blue. Arrow points to lymphatic vessel and arrowhead to lymph node. Lymphatic function was measured by quantification of Evans Blue in the sera of Apoe −/− (white bar) and Panx1 −/− Apoe −/− (grey bar) mice ( D ; n = 4), and of Panx1 fl/fl Apoe −/− (white bar) and Panx1 del Apoe −/− (black bar) mice ( E ; n = 6). ( F ) Representative images of Hematoxylin/Eosin stained cryosections of tails (1 cm from top) from Apoe −/− and Panx1 −/− Apoe −/− mice. Asterisks denote regions rich in microvasculature. Scale bar represents 100 μm. Tail diameter quantification in Apoe −/− (white bars) and Panx1 −/− Apoe −/− (grey bars) mice was measured at ( G ) 1 cm from the basis and at ( H ) 4 cm from the tip of the tail (n = 16–19). ( I ) LYVE-1 immunostaining (red; arrows) in intestinal villi of Apoe −/− and Panx1 −/− Apoe −/− mice. Nuclei were stained with DAPI (blue). Scale bar represents 50 μm. TG ( J ) and FFA ( K ) concentration measured before and 3 hours after olive oil gavage of Apoe −/− (white bars) and Panx1 −/− Apoe −/− (grey bars) mice (n = 6).
    Figure Legend Snippet: Panx1 deficiency reduces lymphatic function. ( A ) Panx1 expression in aortic ECs and LECs from WT mice was assessed by real-time qPCR (n = 3–4). ( B ) Percentage of resident and migratory DCs in CH draining lymph nodes of control (white bars) and Panx1 −/− mice (grey bars) (n = 5). ( C ) Representative images of lymphatic drainage 1 and 15 min after injection of 5 μl of Evans Blue. Arrow points to lymphatic vessel and arrowhead to lymph node. Lymphatic function was measured by quantification of Evans Blue in the sera of Apoe −/− (white bar) and Panx1 −/− Apoe −/− (grey bar) mice ( D ; n = 4), and of Panx1 fl/fl Apoe −/− (white bar) and Panx1 del Apoe −/− (black bar) mice ( E ; n = 6). ( F ) Representative images of Hematoxylin/Eosin stained cryosections of tails (1 cm from top) from Apoe −/− and Panx1 −/− Apoe −/− mice. Asterisks denote regions rich in microvasculature. Scale bar represents 100 μm. Tail diameter quantification in Apoe −/− (white bars) and Panx1 −/− Apoe −/− (grey bars) mice was measured at ( G ) 1 cm from the basis and at ( H ) 4 cm from the tip of the tail (n = 16–19). ( I ) LYVE-1 immunostaining (red; arrows) in intestinal villi of Apoe −/− and Panx1 −/− Apoe −/− mice. Nuclei were stained with DAPI (blue). Scale bar represents 50 μm. TG ( J ) and FFA ( K ) concentration measured before and 3 hours after olive oil gavage of Apoe −/− (white bars) and Panx1 −/− Apoe −/− (grey bars) mice (n = 6).

    Techniques Used: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction, Injection, Staining, Immunostaining, Concentration Assay

    Targeted deletion of Panx1 in endothelial and monocytic cells favors atherosclerotic plaque development. Representative images of Panx1 immunofluorescent staining in ECs of a mouse carotid artery ( A and B ) and in macrophage foam cells within a carotid atherosclerotic lesion ( C and D ). Nuclei were stained with DAPI (blue) and elastic laminae were visualized with Evans Blue (red). Scale bars represent 50 or 25 μm, respectively. Panx1 expression in ECs ( E ) and BMDMs ( F ) of Panx1 fl/ fl Apoe −/− (white bars) and Panx1 del Apoe −/− (black bars) mice was assessed by real-time qPCR (n = 6). ( G ) Weights of Panx1 fl/fl Apoe −/− and Panx1 del Apoe −/− mice before and after 10 weeks of HCD (n = 10). Serum total cholesterol ( H ) and TG ( I ) levels in Panx1 fl/fl Apoe −/− and Panx1 del Apoe −/− mice after 10 weeks of HCD (n = 10). Sudan-IV staining ( J ) and quantification of atherosclerotic lesion extent ( K ) in the thoracic-abdominal aortas and the aortic roots ( L and M ) of Panx1 fl/fl Apoe −/− and Panx1 del Apoe −/− mice after 10 weeks of HCD (n = 10). Scale bar represents 200 μm.
    Figure Legend Snippet: Targeted deletion of Panx1 in endothelial and monocytic cells favors atherosclerotic plaque development. Representative images of Panx1 immunofluorescent staining in ECs of a mouse carotid artery ( A and B ) and in macrophage foam cells within a carotid atherosclerotic lesion ( C and D ). Nuclei were stained with DAPI (blue) and elastic laminae were visualized with Evans Blue (red). Scale bars represent 50 or 25 μm, respectively. Panx1 expression in ECs ( E ) and BMDMs ( F ) of Panx1 fl/ fl Apoe −/− (white bars) and Panx1 del Apoe −/− (black bars) mice was assessed by real-time qPCR (n = 6). ( G ) Weights of Panx1 fl/fl Apoe −/− and Panx1 del Apoe −/− mice before and after 10 weeks of HCD (n = 10). Serum total cholesterol ( H ) and TG ( I ) levels in Panx1 fl/fl Apoe −/− and Panx1 del Apoe −/− mice after 10 weeks of HCD (n = 10). Sudan-IV staining ( J ) and quantification of atherosclerotic lesion extent ( K ) in the thoracic-abdominal aortas and the aortic roots ( L and M ) of Panx1 fl/fl Apoe −/− and Panx1 del Apoe −/− mice after 10 weeks of HCD (n = 10). Scale bar represents 200 μm.

    Techniques Used: Staining, Expressing, Mouse Assay, Real-time Polymerase Chain Reaction

    Ubiquitous Panx1 deletion does not affect atherogenesis. Sudan-IV staining ( A ) and quantification of atherosclerotic lesion extent in the thoracic-abdominal aortas ( B ) and in the aortic roots ( C ) of Apoe −/− (white bars) and Panx1 −/− Apoe −/− (grey bars) mice after 5 weeks of HCD (n = 10). Scale bar represents 200 μm. ( D ) Representative image illustrating the difference in size between Apoe −/− and Panx1 −/− Apoe −/− mice at the age of 10 weeks (n = 10). ( E ) Weights of Apoe −/− and Panx1 −/− Apoe −/− mice before and after 5 weeks of HCD (n = 10). Fat mass ( F ) and lean mass ( G ) of Apoe −/− and Panx1 −/− Apoe −/− mice were measured by MRI (n = 5). Subcutaneous adipose tissue (SAT; H ) and visceral adipose tissue (VAT; I ) in Apoe −/− and Panx1 −/− Apoe −/− mice was determined by micro X-ray computed tomography (n = 10). Serum total cholesterol ( J ), TG ( K ), FFA ( L ), LDL ( M ) and HDL ( N ) levels in Apoe −/− and Panx1 −/− Apoe −/− mice were measured after 5 weeks of HCD (n = 10).
    Figure Legend Snippet: Ubiquitous Panx1 deletion does not affect atherogenesis. Sudan-IV staining ( A ) and quantification of atherosclerotic lesion extent in the thoracic-abdominal aortas ( B ) and in the aortic roots ( C ) of Apoe −/− (white bars) and Panx1 −/− Apoe −/− (grey bars) mice after 5 weeks of HCD (n = 10). Scale bar represents 200 μm. ( D ) Representative image illustrating the difference in size between Apoe −/− and Panx1 −/− Apoe −/− mice at the age of 10 weeks (n = 10). ( E ) Weights of Apoe −/− and Panx1 −/− Apoe −/− mice before and after 5 weeks of HCD (n = 10). Fat mass ( F ) and lean mass ( G ) of Apoe −/− and Panx1 −/− Apoe −/− mice were measured by MRI (n = 5). Subcutaneous adipose tissue (SAT; H ) and visceral adipose tissue (VAT; I ) in Apoe −/− and Panx1 −/− Apoe −/− mice was determined by micro X-ray computed tomography (n = 10). Serum total cholesterol ( J ), TG ( K ), FFA ( L ), LDL ( M ) and HDL ( N ) levels in Apoe −/− and Panx1 −/− Apoe −/− mice were measured after 5 weeks of HCD (n = 10).

    Techniques Used: Staining, Mouse Assay, Magnetic Resonance Imaging, Computed Tomography

    12) Product Images from "Nicotine protects rat hypoglossal motoneurons from excitotoxic death via downregulation of connexin 36"

    Article Title: Nicotine protects rat hypoglossal motoneurons from excitotoxic death via downregulation of connexin 36

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2017.232

    Panx1 expression remains unchanged. ( a ) Example of HMs after 4-h incubation in Krebs (sham), TBOA, nicotine + TBOA, or nicotine, labeled with the neuronal marker SMI 32 (left column; green), or Panx1 (middle column; red). Merged images are shown on the right column where DAPI (blue) is used as nuclear marker. ( b ) Histograms quantifying unchanged levels of Panx1 among the treatments described above. ( c ) Example of western immunoblotting (top) showing the unchanged expression of Panx1 in brainstems incubated as described above
    Figure Legend Snippet: Panx1 expression remains unchanged. ( a ) Example of HMs after 4-h incubation in Krebs (sham), TBOA, nicotine + TBOA, or nicotine, labeled with the neuronal marker SMI 32 (left column; green), or Panx1 (middle column; red). Merged images are shown on the right column where DAPI (blue) is used as nuclear marker. ( b ) Histograms quantifying unchanged levels of Panx1 among the treatments described above. ( c ) Example of western immunoblotting (top) showing the unchanged expression of Panx1 in brainstems incubated as described above

    Techniques Used: Expressing, Incubation, Labeling, Marker, Western Blot

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    Alomone Labs rabbit anti panx1 antibody
    Cholesterol depletion reduces lateral diffusion of mPanx-1-GFP in <t>Panx1-null</t> astrocytes. ( A ) <t>Panx1-null</t> astrocytes transiently expressing mPanx-1-GFP were subjected to treatments with 5 mM methyl-β-cyclodextrin (MβCD), 5 mM MβCD followed by addition of cholesterol (MβCD + Chol) or left untreated (CTRL). Typical FRAP curves of the lateral diffusion of mPanx1-GFP and its modulation by the various treatments are depicted in A. mPanx1-GFP lateral diffusion rates were reduced by MβCD ( B ). Normal diffusion was restored when cholesterol was added. (* p
    Rabbit Anti Panx1 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cholesterol depletion reduces lateral diffusion of mPanx-1-GFP in Panx1-null astrocytes. ( A ) Panx1-null astrocytes transiently expressing mPanx-1-GFP were subjected to treatments with 5 mM methyl-β-cyclodextrin (MβCD), 5 mM MβCD followed by addition of cholesterol (MβCD + Chol) or left untreated (CTRL). Typical FRAP curves of the lateral diffusion of mPanx1-GFP and its modulation by the various treatments are depicted in A. mPanx1-GFP lateral diffusion rates were reduced by MβCD ( B ). Normal diffusion was restored when cholesterol was added. (* p

    Journal: Cells

    Article Title: Activity and Stability of Panx1 Channels in Astrocytes and Neuroblastoma Cells Are Enhanced by Cholesterol Depletion

    doi: 10.3390/cells11203219

    Figure Lengend Snippet: Cholesterol depletion reduces lateral diffusion of mPanx-1-GFP in Panx1-null astrocytes. ( A ) Panx1-null astrocytes transiently expressing mPanx-1-GFP were subjected to treatments with 5 mM methyl-β-cyclodextrin (MβCD), 5 mM MβCD followed by addition of cholesterol (MβCD + Chol) or left untreated (CTRL). Typical FRAP curves of the lateral diffusion of mPanx1-GFP and its modulation by the various treatments are depicted in A. mPanx1-GFP lateral diffusion rates were reduced by MβCD ( B ). Normal diffusion was restored when cholesterol was added. (* p

    Article Snippet: Cells fixed in 4% p-formaldehyde (PFA) were permeabilized with 0.2% Triton X-100 for 15 min, then incubated for 30 min in a blocking solution (2% BSA in PBS) and then overnight with rabbit anti-Panx1 antibody (1:200; Alomone, Jerusalem, Israel; cat#ACC-234) in blocking solution.

    Techniques: Diffusion-based Assay, Expressing

    Human Panx1 conductance is activated by cholesterol depletion. ( A , B ) Electrophysiological recordings obtained from N2a cells expressing hPanx1 subjected to treatment with 5 mM MβCD ( A , D ) and 5 µM lovastatin ( B , E ). Note that N2a hPanx-1 transfected cells responded to cholesterol depletion with increased current amplitudes (red and green curves, in A and B, respectively) in response to 5.5 s-long voltage ramps from −60 to +80 mV compared to untreated cells, CTRL (black curves). Mean ± SEM values of the fold changes in peak conductance induced by 5 mM MβCD and 10 µM lovastatin obtained for N2a hPanx1 transfected cells are shown in parts ( C , D ), respectively. p values were obtained using Mann–Whitney tests. n = 10–11 cells. ** p

    Journal: Cells

    Article Title: Activity and Stability of Panx1 Channels in Astrocytes and Neuroblastoma Cells Are Enhanced by Cholesterol Depletion

    doi: 10.3390/cells11203219

    Figure Lengend Snippet: Human Panx1 conductance is activated by cholesterol depletion. ( A , B ) Electrophysiological recordings obtained from N2a cells expressing hPanx1 subjected to treatment with 5 mM MβCD ( A , D ) and 5 µM lovastatin ( B , E ). Note that N2a hPanx-1 transfected cells responded to cholesterol depletion with increased current amplitudes (red and green curves, in A and B, respectively) in response to 5.5 s-long voltage ramps from −60 to +80 mV compared to untreated cells, CTRL (black curves). Mean ± SEM values of the fold changes in peak conductance induced by 5 mM MβCD and 10 µM lovastatin obtained for N2a hPanx1 transfected cells are shown in parts ( C , D ), respectively. p values were obtained using Mann–Whitney tests. n = 10–11 cells. ** p

    Article Snippet: Cells fixed in 4% p-formaldehyde (PFA) were permeabilized with 0.2% Triton X-100 for 15 min, then incubated for 30 min in a blocking solution (2% BSA in PBS) and then overnight with rabbit anti-Panx1 antibody (1:200; Alomone, Jerusalem, Israel; cat#ACC-234) in blocking solution.

    Techniques: Expressing, Transfection, MANN-WHITNEY

    Panx1 channels mediate dye uptake in cholesterol-depleted cell plasma membrane of N2a cells. ( A ) Representative photomicrographs of mPanx1 transfected and Panx1-null. ( C ) N2a cells showing EthBr uptake after MβCD and lovastatin with and without 100 µM BzATP treatment. Scale bar: 100 µm. ( B , D ) Mean ± SEM values of the relative EthBr fluorescence intensity. Values were normalized to those obtained under control conditions. Each point on the graphs represents the mean values of relative EthBr fluorescence changes recorded from all cells present in a field of view obtained from 3–5 independent experiments. p values obtained from ANOVA followed by Dunnett’s test. * p

    Journal: Cells

    Article Title: Activity and Stability of Panx1 Channels in Astrocytes and Neuroblastoma Cells Are Enhanced by Cholesterol Depletion

    doi: 10.3390/cells11203219

    Figure Lengend Snippet: Panx1 channels mediate dye uptake in cholesterol-depleted cell plasma membrane of N2a cells. ( A ) Representative photomicrographs of mPanx1 transfected and Panx1-null. ( C ) N2a cells showing EthBr uptake after MβCD and lovastatin with and without 100 µM BzATP treatment. Scale bar: 100 µm. ( B , D ) Mean ± SEM values of the relative EthBr fluorescence intensity. Values were normalized to those obtained under control conditions. Each point on the graphs represents the mean values of relative EthBr fluorescence changes recorded from all cells present in a field of view obtained from 3–5 independent experiments. p values obtained from ANOVA followed by Dunnett’s test. * p

    Article Snippet: Cells fixed in 4% p-formaldehyde (PFA) were permeabilized with 0.2% Triton X-100 for 15 min, then incubated for 30 min in a blocking solution (2% BSA in PBS) and then overnight with rabbit anti-Panx1 antibody (1:200; Alomone, Jerusalem, Israel; cat#ACC-234) in blocking solution.

    Techniques: Transfection, Fluorescence

    Cholesterol depletion enhances dye uptake through Panx1 channels in astrocytes. ( A ) Fluorescence micrographs of intracellular EthBr in normal conditions (CTRL) and after treatment with MβCD, BzATP, or with both drugs. Scale bar: 100 µm. ( B ) EthBr uptake in Panx1-null astrocytes induced by MβCD with and without BzATP. Scale bar: 100 µm. ( C ) Histograms showing relative EthBr uptake compared to controls for treatment with MβCD alone or together with BzATP. ( D ) Mean ± SEM values of the relative EthBr fluorescence intensity obtained from Panx1-null astrocytes after MβCD, 100 µM BzATP, and MβCD with BzATP treatment. Values were normalized to those obtained under control conditions. In ( C , D ), each bar on the graphs represents mean value of relative EthBr fluorescence changes recorded from all cells present in a field of view obtained from 3–5 independent experiments. p values obtained from ANOVA test followed by Dunnett’s test. *** p

    Journal: Cells

    Article Title: Activity and Stability of Panx1 Channels in Astrocytes and Neuroblastoma Cells Are Enhanced by Cholesterol Depletion

    doi: 10.3390/cells11203219

    Figure Lengend Snippet: Cholesterol depletion enhances dye uptake through Panx1 channels in astrocytes. ( A ) Fluorescence micrographs of intracellular EthBr in normal conditions (CTRL) and after treatment with MβCD, BzATP, or with both drugs. Scale bar: 100 µm. ( B ) EthBr uptake in Panx1-null astrocytes induced by MβCD with and without BzATP. Scale bar: 100 µm. ( C ) Histograms showing relative EthBr uptake compared to controls for treatment with MβCD alone or together with BzATP. ( D ) Mean ± SEM values of the relative EthBr fluorescence intensity obtained from Panx1-null astrocytes after MβCD, 100 µM BzATP, and MβCD with BzATP treatment. Values were normalized to those obtained under control conditions. In ( C , D ), each bar on the graphs represents mean value of relative EthBr fluorescence changes recorded from all cells present in a field of view obtained from 3–5 independent experiments. p values obtained from ANOVA test followed by Dunnett’s test. *** p

    Article Snippet: Cells fixed in 4% p-formaldehyde (PFA) were permeabilized with 0.2% Triton X-100 for 15 min, then incubated for 30 min in a blocking solution (2% BSA in PBS) and then overnight with rabbit anti-Panx1 antibody (1:200; Alomone, Jerusalem, Israel; cat#ACC-234) in blocking solution.

    Techniques: Fluorescence

    Panx1 channels mediate ATP release from cultured cholesterol depleted astrocytes. Histograms showing the ATP release from cultured Panx1 f/f and Panx1-null astrocytes induced by cholesterol depletion with 5 mM MβCD ( A , B ) and 10 µM lovastatin ( C , D ). Bar histogram of the mean ± SEM values of ATP release from cultured Panx1 f/f and Panx1 null astrocytes induced by cholesterol depletion. p values were obtained using Mann–Whitney tests. Data are from 2–3 independent experiments. * p

    Journal: Cells

    Article Title: Activity and Stability of Panx1 Channels in Astrocytes and Neuroblastoma Cells Are Enhanced by Cholesterol Depletion

    doi: 10.3390/cells11203219

    Figure Lengend Snippet: Panx1 channels mediate ATP release from cultured cholesterol depleted astrocytes. Histograms showing the ATP release from cultured Panx1 f/f and Panx1-null astrocytes induced by cholesterol depletion with 5 mM MβCD ( A , B ) and 10 µM lovastatin ( C , D ). Bar histogram of the mean ± SEM values of ATP release from cultured Panx1 f/f and Panx1 null astrocytes induced by cholesterol depletion. p values were obtained using Mann–Whitney tests. Data are from 2–3 independent experiments. * p

    Article Snippet: Cells fixed in 4% p-formaldehyde (PFA) were permeabilized with 0.2% Triton X-100 for 15 min, then incubated for 30 min in a blocking solution (2% BSA in PBS) and then overnight with rabbit anti-Panx1 antibody (1:200; Alomone, Jerusalem, Israel; cat#ACC-234) in blocking solution.

    Techniques: Cell Culture, MANN-WHITNEY

    Effects of treatment with MβCD and lovastatin on membrane cholesterol level, Panx1 extraction, and cell viability. ( A ) mPanx-1-GFP transfected N2a cells were incubated with cyclodextrin (5 mM MβCD and 10 mM MβCD for 60 min at 37 °C), lovastatin (5 μM, 12 h) in serum-free DMEM or with cholesterol (2 h, 37 °C). Washed membrane fractions were assayed for cholesterol with results normalized with respect to the protein content of the membrane fraction. Bars show mean ± SEM of replicates of three experiments. ( B ) Effects of MβCD and lovastatin in medium of Panx1 cells. ( C ) Effects of MβCD, lovastatin, and cholesterol on cell viability of mPanx1-GFP transfected N2a cells measured by MTT assay after treatments. p values obtained from ANOVA test followed by Dunnett’s test. Results are representative of three independent experiments ± SEM.* p

    Journal: Cells

    Article Title: Activity and Stability of Panx1 Channels in Astrocytes and Neuroblastoma Cells Are Enhanced by Cholesterol Depletion

    doi: 10.3390/cells11203219

    Figure Lengend Snippet: Effects of treatment with MβCD and lovastatin on membrane cholesterol level, Panx1 extraction, and cell viability. ( A ) mPanx-1-GFP transfected N2a cells were incubated with cyclodextrin (5 mM MβCD and 10 mM MβCD for 60 min at 37 °C), lovastatin (5 μM, 12 h) in serum-free DMEM or with cholesterol (2 h, 37 °C). Washed membrane fractions were assayed for cholesterol with results normalized with respect to the protein content of the membrane fraction. Bars show mean ± SEM of replicates of three experiments. ( B ) Effects of MβCD and lovastatin in medium of Panx1 cells. ( C ) Effects of MβCD, lovastatin, and cholesterol on cell viability of mPanx1-GFP transfected N2a cells measured by MTT assay after treatments. p values obtained from ANOVA test followed by Dunnett’s test. Results are representative of three independent experiments ± SEM.* p

    Article Snippet: Cells fixed in 4% p-formaldehyde (PFA) were permeabilized with 0.2% Triton X-100 for 15 min, then incubated for 30 min in a blocking solution (2% BSA in PBS) and then overnight with rabbit anti-Panx1 antibody (1:200; Alomone, Jerusalem, Israel; cat#ACC-234) in blocking solution.

    Techniques: Transfection, Incubation, MTT Assay

    Cholesterol depletion reduces lateral mobility of Panx1 in the membrane. N2a cells transiently expressing mPanx-1-GFP were subjected to treatment with 5 mM methyl-β-cyclodextrin (MβCD), 5 mM MβCD followed by addition of cholesterol (MβCD + Chol) or left untreated (CTRL). ( A – C ) Fluorescence images of transfected cells (control and treated with MβCD alone or with cholesterol) before (pre-bleach) and at 0.6 and 60 s postbleach; bleached regions are shown at higher magnification in insets. Bars: 10 μm; inset magnification 3×. ( D ) FRAP recovery curves for control cells and with MβCD alone or with cholesterol. ( E ) Histograms of normalized FRAP recovery at 15, 30, and 60 s after photobleach. p values obtained from ANOVA test followed by Dunnett’s test. n = 7–10 per plasma membrane domain, data collected over four independent repeats. ** p

    Journal: Cells

    Article Title: Activity and Stability of Panx1 Channels in Astrocytes and Neuroblastoma Cells Are Enhanced by Cholesterol Depletion

    doi: 10.3390/cells11203219

    Figure Lengend Snippet: Cholesterol depletion reduces lateral mobility of Panx1 in the membrane. N2a cells transiently expressing mPanx-1-GFP were subjected to treatment with 5 mM methyl-β-cyclodextrin (MβCD), 5 mM MβCD followed by addition of cholesterol (MβCD + Chol) or left untreated (CTRL). ( A – C ) Fluorescence images of transfected cells (control and treated with MβCD alone or with cholesterol) before (pre-bleach) and at 0.6 and 60 s postbleach; bleached regions are shown at higher magnification in insets. Bars: 10 μm; inset magnification 3×. ( D ) FRAP recovery curves for control cells and with MβCD alone or with cholesterol. ( E ) Histograms of normalized FRAP recovery at 15, 30, and 60 s after photobleach. p values obtained from ANOVA test followed by Dunnett’s test. n = 7–10 per plasma membrane domain, data collected over four independent repeats. ** p

    Article Snippet: Cells fixed in 4% p-formaldehyde (PFA) were permeabilized with 0.2% Triton X-100 for 15 min, then incubated for 30 min in a blocking solution (2% BSA in PBS) and then overnight with rabbit anti-Panx1 antibody (1:200; Alomone, Jerusalem, Israel; cat#ACC-234) in blocking solution.

    Techniques: Expressing, Fluorescence, Transfection

    Expression of endogenous Panx1 is largely absent in CRISPR-deleted N2a cells. Representative confocal images obtained from ( A ) parental, ( B ) Panx1-deleted, and ( C ) Panx1-deleted N2a cells expressing human (h) Panx1 construct. Boxed white areas show the Panx1 staining at higher magnification. Scale bars: 50 μm lower magnification, 20 μm in boxed areas.

    Journal: Cells

    Article Title: Activity and Stability of Panx1 Channels in Astrocytes and Neuroblastoma Cells Are Enhanced by Cholesterol Depletion

    doi: 10.3390/cells11203219

    Figure Lengend Snippet: Expression of endogenous Panx1 is largely absent in CRISPR-deleted N2a cells. Representative confocal images obtained from ( A ) parental, ( B ) Panx1-deleted, and ( C ) Panx1-deleted N2a cells expressing human (h) Panx1 construct. Boxed white areas show the Panx1 staining at higher magnification. Scale bars: 50 μm lower magnification, 20 μm in boxed areas.

    Article Snippet: Cells fixed in 4% p-formaldehyde (PFA) were permeabilized with 0.2% Triton X-100 for 15 min, then incubated for 30 min in a blocking solution (2% BSA in PBS) and then overnight with rabbit anti-Panx1 antibody (1:200; Alomone, Jerusalem, Israel; cat#ACC-234) in blocking solution.

    Techniques: Expressing, CRISPR, Construct, Staining

    TO-PRO-3 uptake in viable and non-apoptotic stom + and stom − K562 cells after hypotonic stress. ( A ) Expression of PANX1 in stom − and stom + K562 clones was assessed by flow cytometry, giving similar levels. ( B ) Comparison of the relative TO-PRO-3 fluorescence intensity (F − F0/F0 − 100) between stom + (black squares) and stom − (grey squares) K562 cell clones using an unpaired t-test. ( C ) Cell volume of stom + (black) and stom − (grey) K562 clones assessed by flow cytometry comparing FSC geomean values obtained in isotonic (circles) and hypotonic (80 mosm/kg, squares) conditions. ( D ) Caspase 3/7 activity measured by flow cytometry analysis allowing the determination of the percentage of fluorescent stom + (iso: black and hypo: black with white dots) and stom − (iso: grey and hypo: grey with white dots) K562 cell clones preloaded by the NucView substrate and stimulated by hypotonic stress (two-way ANOVA Sidak’s multiple comparisons test). ( p values > 0.05: ns, ≤0.05: *, ≤0.01: ** and ≤0.0001: ****).

    Journal: International Journal of Molecular Sciences

    Article Title: Mechanosensitive Pannexin 1 Activity Is Modulated by Stomatin in Human Red Blood Cells

    doi: 10.3390/ijms23169401

    Figure Lengend Snippet: TO-PRO-3 uptake in viable and non-apoptotic stom + and stom − K562 cells after hypotonic stress. ( A ) Expression of PANX1 in stom − and stom + K562 clones was assessed by flow cytometry, giving similar levels. ( B ) Comparison of the relative TO-PRO-3 fluorescence intensity (F − F0/F0 − 100) between stom + (black squares) and stom − (grey squares) K562 cell clones using an unpaired t-test. ( C ) Cell volume of stom + (black) and stom − (grey) K562 clones assessed by flow cytometry comparing FSC geomean values obtained in isotonic (circles) and hypotonic (80 mosm/kg, squares) conditions. ( D ) Caspase 3/7 activity measured by flow cytometry analysis allowing the determination of the percentage of fluorescent stom + (iso: black and hypo: black with white dots) and stom − (iso: grey and hypo: grey with white dots) K562 cell clones preloaded by the NucView substrate and stimulated by hypotonic stress (two-way ANOVA Sidak’s multiple comparisons test). ( p values > 0.05: ns, ≤0.05: *, ≤0.01: ** and ≤0.0001: ****).

    Article Snippet: Proteins were stained with 1/200 mouse anti-Stomatin E5 monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA) or 1/100 rabbit anti-PANX1 polyclonal antibody (Alomone Labs, Jerusalem, Israel) in DPBS-BSA 0.2% for 1 h at 37 °C.

    Techniques: Expressing, Clone Assay, Flow Cytometry, Fluorescence, Activity Assay

    Endogenous expression of stomatin and PANX1 in K562 cells and PANX1-dependent TO-PRO-3 uptake by non-apoptotic K562 cells. ( A ) Stomatin expression analysed by flow cytometry on permeabilized K562 cells using anti-stomatin E5 (Santa Cruz Biotechnology, Dallas, TX, USA). ( B ) PANX1 expression analysed by flow cytometry on permeabilized K562 cells using anti-PANX1 (Alomone Labs, Jerusalem, Israel). ( C ) Gating strategy of Sytox-negative (Pacific Blue) and Lactadherin-negative (FITC) K562 cells (at least 85%, Q4) in which the TO-PRO-3 staining (APC) was evaluated by flow cytometry. ( D ) TO-PRO-3 uptake was measured on K562 cells in basal conditions and, after a hypotonic stimulation (80 mOsm/kg), on K562 cells that were preincubated or not with several PANX1 inhibitors (carbenoxolone [CBX], probenecid [PBC], and mefloquine [MFQ]) (N = 3).

    Journal: International Journal of Molecular Sciences

    Article Title: Mechanosensitive Pannexin 1 Activity Is Modulated by Stomatin in Human Red Blood Cells

    doi: 10.3390/ijms23169401

    Figure Lengend Snippet: Endogenous expression of stomatin and PANX1 in K562 cells and PANX1-dependent TO-PRO-3 uptake by non-apoptotic K562 cells. ( A ) Stomatin expression analysed by flow cytometry on permeabilized K562 cells using anti-stomatin E5 (Santa Cruz Biotechnology, Dallas, TX, USA). ( B ) PANX1 expression analysed by flow cytometry on permeabilized K562 cells using anti-PANX1 (Alomone Labs, Jerusalem, Israel). ( C ) Gating strategy of Sytox-negative (Pacific Blue) and Lactadherin-negative (FITC) K562 cells (at least 85%, Q4) in which the TO-PRO-3 staining (APC) was evaluated by flow cytometry. ( D ) TO-PRO-3 uptake was measured on K562 cells in basal conditions and, after a hypotonic stimulation (80 mOsm/kg), on K562 cells that were preincubated or not with several PANX1 inhibitors (carbenoxolone [CBX], probenecid [PBC], and mefloquine [MFQ]) (N = 3).

    Article Snippet: Proteins were stained with 1/200 mouse anti-Stomatin E5 monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA) or 1/100 rabbit anti-PANX1 polyclonal antibody (Alomone Labs, Jerusalem, Israel) in DPBS-BSA 0.2% for 1 h at 37 °C.

    Techniques: Expressing, Flow Cytometry, Staining

    Activation of ATP release in frozen/thawed and rejuvenated RBCs from controls and OHSt patients. Suspensions of frozen/thawed and rejuvenated RBCs were exposed to isoosmotic (Iso) or hypoosmotic (Hypo, 240 mOsm/kg) stimulation. Experiments were performed with or without preincubation with 100 μM CBX, a PANX1 inhibitor. Results are expressed as eATP (pmol/10 6 cells). ( A ) Stacked graph showing total eATP including lytic eATP (hatched bars) released from control (white bars) or OHSt (grey bars) RBCs. The hemoglobin concentration of paired samples was used to estimate lytic eATP. ( B ) Non-lytic eATP was calculated as the differences between total eATP and lytic eATP. Total, lytic, and non-lytic eATP values were compared using a One-Way ANOVA Dunnett’s test for Multiple Comparison (N = 3). The results shown are mean values ± SEM. ( p values > 0.05: ns, ≤0.05: *, ≤0.01: **). ns stands for not significant.

    Journal: International Journal of Molecular Sciences

    Article Title: Mechanosensitive Pannexin 1 Activity Is Modulated by Stomatin in Human Red Blood Cells

    doi: 10.3390/ijms23169401

    Figure Lengend Snippet: Activation of ATP release in frozen/thawed and rejuvenated RBCs from controls and OHSt patients. Suspensions of frozen/thawed and rejuvenated RBCs were exposed to isoosmotic (Iso) or hypoosmotic (Hypo, 240 mOsm/kg) stimulation. Experiments were performed with or without preincubation with 100 μM CBX, a PANX1 inhibitor. Results are expressed as eATP (pmol/10 6 cells). ( A ) Stacked graph showing total eATP including lytic eATP (hatched bars) released from control (white bars) or OHSt (grey bars) RBCs. The hemoglobin concentration of paired samples was used to estimate lytic eATP. ( B ) Non-lytic eATP was calculated as the differences between total eATP and lytic eATP. Total, lytic, and non-lytic eATP values were compared using a One-Way ANOVA Dunnett’s test for Multiple Comparison (N = 3). The results shown are mean values ± SEM. ( p values > 0.05: ns, ≤0.05: *, ≤0.01: **). ns stands for not significant.

    Article Snippet: Proteins were stained with 1/200 mouse anti-Stomatin E5 monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA) or 1/100 rabbit anti-PANX1 polyclonal antibody (Alomone Labs, Jerusalem, Israel) in DPBS-BSA 0.2% for 1 h at 37 °C.

    Techniques: Activation Assay, Concentration Assay

    5(6)-carboxyfluorescein uptake in control (Ctrl) and OHSt RBCs after stimulation by high K + . ( A ) MFI due to the dye uptake in Ctrl (black) and OHSt (grey) frozen/thawed rejuvenated RBCs measured by flow cytometry in basal conditions and after stimulation by KCl without or with incubation with CBX. ( B ) Carbenoxolone (CBX), probenecid (PBC), and mefloquine (MFQ), three different PANX1 inhibitors, were added to RBC 20 min before the stimulation, resulting in a fluorescence signal similar to that of basal conditions. ( C ) FSC was also determined in all the conditions described above under KCl stimulation. Data obtained from stimulated cells were compared to that of unstimulated cells or cells stimulated in the presence of CBX using a paired Two-way Anova Sidak’s multiple comparisons test. The same test but unpaired was used when Ctrl and OHSt RBCs were compared. ( p values > 0.05: ns, ≤0.05: *, ≤0.0001: ****). ns stands for not significant.

    Journal: International Journal of Molecular Sciences

    Article Title: Mechanosensitive Pannexin 1 Activity Is Modulated by Stomatin in Human Red Blood Cells

    doi: 10.3390/ijms23169401

    Figure Lengend Snippet: 5(6)-carboxyfluorescein uptake in control (Ctrl) and OHSt RBCs after stimulation by high K + . ( A ) MFI due to the dye uptake in Ctrl (black) and OHSt (grey) frozen/thawed rejuvenated RBCs measured by flow cytometry in basal conditions and after stimulation by KCl without or with incubation with CBX. ( B ) Carbenoxolone (CBX), probenecid (PBC), and mefloquine (MFQ), three different PANX1 inhibitors, were added to RBC 20 min before the stimulation, resulting in a fluorescence signal similar to that of basal conditions. ( C ) FSC was also determined in all the conditions described above under KCl stimulation. Data obtained from stimulated cells were compared to that of unstimulated cells or cells stimulated in the presence of CBX using a paired Two-way Anova Sidak’s multiple comparisons test. The same test but unpaired was used when Ctrl and OHSt RBCs were compared. ( p values > 0.05: ns, ≤0.05: *, ≤0.0001: ****). ns stands for not significant.

    Article Snippet: Proteins were stained with 1/200 mouse anti-Stomatin E5 monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA) or 1/100 rabbit anti-PANX1 polyclonal antibody (Alomone Labs, Jerusalem, Israel) in DPBS-BSA 0.2% for 1 h at 37 °C.

    Techniques: Flow Cytometry, Incubation, Fluorescence

    Activation of ATP release in fresh RBCs by a hypoosmotic challenge. ( A ) RBC suspensions were exposed to isoosmotic (Iso) or hypoosmotic (Hypo, 240 mOsm/kg) stimulation. Experiments were performed with or without preincubation with 100 μM CBX, a PANX1 inhibitor. Results are expressed as eATP (pmol/10 6 cells). The hemoglobin concentration of paired samples was used to estimate lytic eATP. A stacked graph was built to show the contribution of lytic ATP (hatched bars) within total ATP release from RBCs. Lytic and total eATP values were compared using a One-Way ANOVA Dunnett’s test for Multiple Comparisons (N = 5). The results shown are mean values ± SEM. ( B ) The difference between total eATP and lytic eATP was calculated and denoted as non-lytic eATP. Non-lytic eATP values were compared using a One-Way ANOVA Dunnett’s test for Multiple Comparisons. ( C ) PS exposure was measured by flow cytometry on Lactadherin-FITC stained RBCs in basal conditions after treatment with Fenton’s reagent, as a control of eryptosis, and in basal or hypoosmotic conditions without Fenton. ( D ) Caspase activation was measured under conditions similar to PS exposure but on FLICA 660 caspase3/7-stained RBCs. The statistical analysis of the last two measurements (N = 5) was performed using an RM-one-way ANOVA test. ( p values > 0.05: ns, ≤0.05: *, ≤0.01: ** and ≤0.001: ***). ns stands for not significant.

    Journal: International Journal of Molecular Sciences

    Article Title: Mechanosensitive Pannexin 1 Activity Is Modulated by Stomatin in Human Red Blood Cells

    doi: 10.3390/ijms23169401

    Figure Lengend Snippet: Activation of ATP release in fresh RBCs by a hypoosmotic challenge. ( A ) RBC suspensions were exposed to isoosmotic (Iso) or hypoosmotic (Hypo, 240 mOsm/kg) stimulation. Experiments were performed with or without preincubation with 100 μM CBX, a PANX1 inhibitor. Results are expressed as eATP (pmol/10 6 cells). The hemoglobin concentration of paired samples was used to estimate lytic eATP. A stacked graph was built to show the contribution of lytic ATP (hatched bars) within total ATP release from RBCs. Lytic and total eATP values were compared using a One-Way ANOVA Dunnett’s test for Multiple Comparisons (N = 5). The results shown are mean values ± SEM. ( B ) The difference between total eATP and lytic eATP was calculated and denoted as non-lytic eATP. Non-lytic eATP values were compared using a One-Way ANOVA Dunnett’s test for Multiple Comparisons. ( C ) PS exposure was measured by flow cytometry on Lactadherin-FITC stained RBCs in basal conditions after treatment with Fenton’s reagent, as a control of eryptosis, and in basal or hypoosmotic conditions without Fenton. ( D ) Caspase activation was measured under conditions similar to PS exposure but on FLICA 660 caspase3/7-stained RBCs. The statistical analysis of the last two measurements (N = 5) was performed using an RM-one-way ANOVA test. ( p values > 0.05: ns, ≤0.05: *, ≤0.01: ** and ≤0.001: ***). ns stands for not significant.

    Article Snippet: Proteins were stained with 1/200 mouse anti-Stomatin E5 monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA) or 1/100 rabbit anti-PANX1 polyclonal antibody (Alomone Labs, Jerusalem, Israel) in DPBS-BSA 0.2% for 1 h at 37 °C.

    Techniques: Activation Assay, Concentration Assay, Flow Cytometry, Staining

    Stomatin and PANX1 proximity in control and OHSt RBCs assessed by Proximity Ligation Assay (PLA) and analyzed by Imaging Flow Cytometry. ( A ) PLA-positive (PLA+) events characterized by red spots which result from the detection oligos coupled to fluorochromes (FarRed) hybridizing to sequences in the amplified DNA were visualized by Imaging Flow Cytometry and merged with Brightfield images of the corresponding Control RBCs. ( B ) Quantification of the assay corresponding to the percentage of PLA+ RBCs was performed several times in different conditions: without any primary antibody (none, N = 10), with only one antibody (anti-stomatin or anti-PANX1 or anti-AE1, N = 3 for each condition) and in the presence of two antibodies (anti-stomatin + anti-PANX1 or anti-stomatin + anti-AE1, N = 7 in both conditions). A comparison was performed using an unpaired One-way Anova Tukey’s test. ( C ) PLA using anti-stomatin + anti-PANX1 was performed in identical conditions to those described above but on frozen/thawed control (Ctrl) and OHSt RBCs and compared using paired or unpaired (between control and OHSt) Two-way Anova Sidak’s multiple comparisons test ( p values > 0.05: ns, ≤0.05: *, ≤0.01: ** and ≤0.001: ***). ns stands for not significant.

    Journal: International Journal of Molecular Sciences

    Article Title: Mechanosensitive Pannexin 1 Activity Is Modulated by Stomatin in Human Red Blood Cells

    doi: 10.3390/ijms23169401

    Figure Lengend Snippet: Stomatin and PANX1 proximity in control and OHSt RBCs assessed by Proximity Ligation Assay (PLA) and analyzed by Imaging Flow Cytometry. ( A ) PLA-positive (PLA+) events characterized by red spots which result from the detection oligos coupled to fluorochromes (FarRed) hybridizing to sequences in the amplified DNA were visualized by Imaging Flow Cytometry and merged with Brightfield images of the corresponding Control RBCs. ( B ) Quantification of the assay corresponding to the percentage of PLA+ RBCs was performed several times in different conditions: without any primary antibody (none, N = 10), with only one antibody (anti-stomatin or anti-PANX1 or anti-AE1, N = 3 for each condition) and in the presence of two antibodies (anti-stomatin + anti-PANX1 or anti-stomatin + anti-AE1, N = 7 in both conditions). A comparison was performed using an unpaired One-way Anova Tukey’s test. ( C ) PLA using anti-stomatin + anti-PANX1 was performed in identical conditions to those described above but on frozen/thawed control (Ctrl) and OHSt RBCs and compared using paired or unpaired (between control and OHSt) Two-way Anova Sidak’s multiple comparisons test ( p values > 0.05: ns, ≤0.05: *, ≤0.01: ** and ≤0.001: ***). ns stands for not significant.

    Article Snippet: Proteins were stained with 1/200 mouse anti-Stomatin E5 monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA) or 1/100 rabbit anti-PANX1 polyclonal antibody (Alomone Labs, Jerusalem, Israel) in DPBS-BSA 0.2% for 1 h at 37 °C.

    Techniques: Proximity Ligation Assay, Imaging, Flow Cytometry, Amplification

    Stomatin and PANX1 expression in OHSt RBCs compared to controls. ( A ) Western blot analysis of RBC ghosts from 3 controls (C1, C2, C2) and the 3 OHSt patients (O1, O2, O3) using mouse monoclonal anti-stomatin (E-5), rabbit polyclonal anti-PANX1 and rabbit polyclonal anti-p55 as control. The molecular weights (kDa) of the detected bands are mentioned. ( B ) Flow cytometry analysis of stomatin expression in the RBCs of the 3 controls and the 3 OHSt patients after permeabilisation. Negative controls consist of the observed fluorescence with only the secondary antibody. The averages of Mean of Fluorescence Intensity (MFI) were compared between OHSt and control (Ctrl) RBCs using a two-way ANOVA Sidak’s multicomparison test ( p values > 0.05: ns and ≤0.05: *, ns stands for not significant), paired for comparisons within OHSt samples when signals with anti-stomatin were compared to that of Negative controls and unpaired when OHSt were compared to Ctrl. ( C ) Flow cytometry analysis of PANX1 expression in the 3 controls and the 3 OHSt patient RBCs after permeabilization and comparisons of MFIs between controls and OHSt, as performed in B.

    Journal: International Journal of Molecular Sciences

    Article Title: Mechanosensitive Pannexin 1 Activity Is Modulated by Stomatin in Human Red Blood Cells

    doi: 10.3390/ijms23169401

    Figure Lengend Snippet: Stomatin and PANX1 expression in OHSt RBCs compared to controls. ( A ) Western blot analysis of RBC ghosts from 3 controls (C1, C2, C2) and the 3 OHSt patients (O1, O2, O3) using mouse monoclonal anti-stomatin (E-5), rabbit polyclonal anti-PANX1 and rabbit polyclonal anti-p55 as control. The molecular weights (kDa) of the detected bands are mentioned. ( B ) Flow cytometry analysis of stomatin expression in the RBCs of the 3 controls and the 3 OHSt patients after permeabilisation. Negative controls consist of the observed fluorescence with only the secondary antibody. The averages of Mean of Fluorescence Intensity (MFI) were compared between OHSt and control (Ctrl) RBCs using a two-way ANOVA Sidak’s multicomparison test ( p values > 0.05: ns and ≤0.05: *, ns stands for not significant), paired for comparisons within OHSt samples when signals with anti-stomatin were compared to that of Negative controls and unpaired when OHSt were compared to Ctrl. ( C ) Flow cytometry analysis of PANX1 expression in the 3 controls and the 3 OHSt patient RBCs after permeabilization and comparisons of MFIs between controls and OHSt, as performed in B.

    Article Snippet: Proteins were stained with 1/200 mouse anti-Stomatin E5 monoclonal antibody (Santa Cruz Biotechnology, Dallas, TX, USA) or 1/100 rabbit anti-PANX1 polyclonal antibody (Alomone Labs, Jerusalem, Israel) in DPBS-BSA 0.2% for 1 h at 37 °C.

    Techniques: Expressing, Western Blot, Flow Cytometry, Fluorescence

    Inhibition of pannexin-1, P2X7, or P2Y2 have different effects on cellular trajectory at the wound edge but all decrease percent wound closure. Confluent Human Corneal Limbal Epithelial cell cultures were pre-incubated in the presence or absence of inhibitors, stained with the SiR Actin, scratch-wounded, and imaged at a rate of one frame per 5 min for 2 h using the Zeiss LSM 880 confocal microscope. (A) Inhibition of pannexin-1, P2X7, or P2Y2 led to significantly diminished wound closure at the 2 h time point. Two-tailed Student’s t-tests were performed to compare the percent wound closure of each inhibitor to an uninhibited control (* p

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Age Dependent Changes in Corneal Epithelial Cell Signaling

    doi: 10.3389/fcell.2022.886721

    Figure Lengend Snippet: Inhibition of pannexin-1, P2X7, or P2Y2 have different effects on cellular trajectory at the wound edge but all decrease percent wound closure. Confluent Human Corneal Limbal Epithelial cell cultures were pre-incubated in the presence or absence of inhibitors, stained with the SiR Actin, scratch-wounded, and imaged at a rate of one frame per 5 min for 2 h using the Zeiss LSM 880 confocal microscope. (A) Inhibition of pannexin-1, P2X7, or P2Y2 led to significantly diminished wound closure at the 2 h time point. Two-tailed Student’s t-tests were performed to compare the percent wound closure of each inhibitor to an uninhibited control (* p

    Article Snippet: Anti-pannexin-1 polyclonal rabbit antibody directed against pannexin-1 (Cat. #ACC-234) was purchased from Alomone Labs (Jerusalem, Israel).

    Techniques: Inhibition, Incubation, Staining, Microscopy, Two Tailed Test

    Localization of pannexin-1 is elevated in corneas from younger mice at the wound edge. Enucleated globes were scratch wounded and allowed to heal for two or 5 h. Globes were fixed in 4% paraformaldehyde, corneas were dissected, and immunohistochemistry was performed to visualize localization of pannexin-1 near the wound edge (green). Imaging was performed using the Zeiss LSM 880 confocal microscope. Topographical maps denoting the intensity of staining throughout the region were generated from the imaging data. (A) In younger mice, pannexin-1 localization is diffuse at 2 h, with similar expression adjacent to and further away from the wound. This is seen in the topographical map as a relatively flat, green diagram. (B) In younger mice at 5 h, the increase in pannexin-1 near the wound edge is detected as red peaks in the topographical map. (C,D) Older mice have a similar localization of pannexin-1 throughout the epithelium at both two and 5 h. The data represent three eyes per condition, with each eye from one mouse. Scale bar is 50 microns.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: Age Dependent Changes in Corneal Epithelial Cell Signaling

    doi: 10.3389/fcell.2022.886721

    Figure Lengend Snippet: Localization of pannexin-1 is elevated in corneas from younger mice at the wound edge. Enucleated globes were scratch wounded and allowed to heal for two or 5 h. Globes were fixed in 4% paraformaldehyde, corneas were dissected, and immunohistochemistry was performed to visualize localization of pannexin-1 near the wound edge (green). Imaging was performed using the Zeiss LSM 880 confocal microscope. Topographical maps denoting the intensity of staining throughout the region were generated from the imaging data. (A) In younger mice, pannexin-1 localization is diffuse at 2 h, with similar expression adjacent to and further away from the wound. This is seen in the topographical map as a relatively flat, green diagram. (B) In younger mice at 5 h, the increase in pannexin-1 near the wound edge is detected as red peaks in the topographical map. (C,D) Older mice have a similar localization of pannexin-1 throughout the epithelium at both two and 5 h. The data represent three eyes per condition, with each eye from one mouse. Scale bar is 50 microns.

    Article Snippet: Anti-pannexin-1 polyclonal rabbit antibody directed against pannexin-1 (Cat. #ACC-234) was purchased from Alomone Labs (Jerusalem, Israel).

    Techniques: Mouse Assay, Immunohistochemistry, Imaging, Microscopy, Staining, Generated, Expressing