Journal: Cell

Article Title: Genomic decoding of neuronal depolarization by stimulus-specific NPAS4 heterodimers

doi: 10.1016/j.cell.2019.09.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse monoclonal anti-ARNTL1 (clone B-1) , Santa Cruz Biotechnology , Cat#sc-365645; RRID:AB_10841724.

Techniques: Virus, Recombinant, RNAscope, Multiplex Assay, Staining, RNA Immunoprecipitation, In Situ, Fluorescence, Derivative Assay, Infection, Library Amplification, Plasmid Preparation, Software

Journal: Developmental cell

Article Title: Canonical Wnt5b signaling directs outlying Nkx2.5+ mesoderm into pacemaker cardiomyocytes

doi: 10.1016/j.devcel.2019.07.014

Figure Lengend Snippet: Key Resources Table

Article Snippet: Chicken anti-GFP antibody , Aves Labs , Cat# GFP-1020.

Techniques: Recombinant, In Situ, RNAscope, Multiplex Assay, SYBR Green Assay, Software

Journal: iScience

Article Title: Pannexin-1 channel opening is critical for COVID-19 pathogenesis

doi: 10.1016/j.isci.2021.103478

Figure Lengend Snippet: SARS-CoV-2 S protein induces Panx-1 channel opening in human lung primary epithelial cells (A–F) Representative snapshots of the time course of Etd uptake in airway epithelial cells for the control at 0, 15, and 30 min. Images shown for each time point are in duplicate with brightfield (left) and fluorescence (right) channels. (G–L) Representative snapshots of the time course after treatment with SARS-CoV-2 S protein (3.5 μg/mL) at 0, 15, and 30 min. Images shown for each time point are in duplicate with brightfield (left) and fluorescence (right) channels. (M) Quantification of the time course of Etd uptake rate from human lung epithelial cells under control conditions (black square) or after SARS-CoV-2 S protein treatment (red circle) for 0 to 60 min. Human lung epithelial cells were pretreated with Probenecid (Prob, blue upward triangle), Panx-1 mimetic peptide (Panx-1pep, green downward triangle), and the scrambled peptide (Scram, pink leftward triangle) for 10 min before the treatment with SARS-CoV-2 S protein. Probenecid and Pannexin-1 mimetic peptide prevented the Panx-1 channel opening induced by SARS-CoV-2 S protein. No significant differences were observed between the control, Prob + S Protein, and Panx-1pep + S protein-treated cells (p = 0.236, for all the points analyzed). No significant differences were observed between S protein and Scram treatments (p = 0.302, for all the points analyzed). For cells treated with S protein alone or S protein pretreated with the scrambled peptide, Scram, all the points from 4 to 24 min were significantly different from control conditions (p = 0.0002 compared to control conditions). Each value corresponds to the mean ± SD of the Etd intracellular intensity present in at least 20 cells per time point (n = 4). (N) Quantification of the time course of Etd uptake rate after treatment of human lung epithelial cells under control (black square) or after hCoV-229E treatment (red circle, 0.1 MOI shown) for 0 to 60 min. The Panx-1 channel opening induced by the 229E virus resulted in a significantly lower uptake rate than cells treated with SARS-CoV-2 S protein alone (p ≤ 1.67x10 −4 , for all the points analyzed). Human lung epithelial cells were pretreated with Probenecid (Prob, blue, upward triangle), Pannexin-1 mimetic peptide (Panx-1pep, green, downward triangle), and the scrambled peptide (Scram, pink leftward triangle) for 10 min before the treatment with hCoV-229E. For hCoV-229E and Scram+229E treatment, all time points, 4 to 12 min, were significantly different from control conditions (p = 0.001 as compared with control conditions). Each value corresponds to the mean ± SD of the Etd intracellular intensity present in at least 20 cells per time point (n = 3).

Article Snippet: SARS-CoV-2 (GISAID, EPI_ISL_406862) and Human Coronavirus 229E (ATCC, VR-740) were propagated in Vero E6 cells.

Techniques: Control, Fluorescence, Virus

Journal: iScience

Article Title: Pannexin-1 channel opening is critical for COVID-19 pathogenesis

doi: 10.1016/j.isci.2021.103478

Figure Lengend Snippet: SARS-CoV-2 S protein induces the opening of Panx-1 channels (A–L) Electrophysiological recordings obtained from human epithelial cells in the absence (black) and presence (red) of (A–C) 1 μM ATP, (D–F) hCoV-229E heat-inactivated (229E, 1 μL/mL) plus 1 μM ATP, (G–I) SARS-CoV-2 S protein (Prot S, 2.5 μg/mL) plus 1 μM ATP, and (J–L) SARS-CoV-2 S protein plus 1 μM ATP in the presence of the Panx-1 channel blocker, Probenecid (Prob, 1.0 mM). (M) Example of currents recorded from an epithelial cell evoked by 12 s long voltage ramps (−70 mV to +70 mV) following the bath application of Prot S and 229E in the presence of ATP (arrows). Note that only SARS-CoV-2 S protein plus 1 μM ATP induced Panx-1 channel openings and that Prob prevented Panx-1 activation. (B, E, H, and K) show the mean ± SEM values of the fold changes in peak conductance measured from epithelial cells under the various conditions, and (C, F, I, and L) show the changes in peak conductance for each individual cell exposed to the experimental conditions. ∗∗∗∗p < 0.0001 (paired t test).

Article Snippet: SARS-CoV-2 (GISAID, EPI_ISL_406862) and Human Coronavirus 229E (ATCC, VR-740) were propagated in Vero E6 cells.

Techniques: Activation Assay

Journal: iScience

Article Title: Pannexin-1 channel opening is critical for COVID-19 pathogenesis

doi: 10.1016/j.isci.2021.103478

Figure Lengend Snippet: Opening Panx-1 channels in response to SARS-CoV-2 S protein or hCoV-229E-virus-induced ATP, PGE 2 , and IL-1β release Upon treating primary lung epithelial cells with SARS-CoV-2 S protein (1 μg/mL) or hCoV-229E (0.1 MOI), media was collected after 1, 6, 12, and 24 h posttreatment to quantify ATP, PGE 2 , and IL-1β. Data after 1 h posttreatment are represented. (A) Determination of ATP secretion from primary human airway epithelial cells for the control (C) and after S protein (S Prot) or 229E virus (229E) treatment in the presence or absence of Probenecid (Pro), Panx-1 mimetic peptide (pep), or the scrambled peptide (Sc). Relative to the control (C), SARS-CoV-2 S protein and hCoV-229E treatment induced an ATP secretion that was strongly Panx-1 dependent. Treatment with the S-protein-induced ATP secretion was ∼2.5-fold more effective than treatment with 229E (∗p ≤ 0.001, n = 4). Pretreatment with Panx-1 blockers, Probenecid, or the Panx-1 mimetic peptide, before treatment with the S Prot or 229E, did not result in significantly elevated concentrations of ATP. In addition, pretreatment with Scram before treatment with S protein or 229E resulted in elevated concentrations of ATP comparable with cells treated with S protein or 229E alone, respectively (∗p ≤ 0.001, n = 4). Each value corresponds to the mean ± SD (n = 4). (B) Determination of PGE 2 secretion from primary human airway epithelial cells for the control (C) and after S protein (S Prot) or hCov-229E virus (229E) treatment in the presence or absence of Probenecid (Pro), Panx-1 mimetic peptide (pep), or the scrambled peptide (Sc). Relative to the control (C), SARS-CoV-2 S protein and hCoV-229E treatment induced a PGE 2 secretion that was also strongly Panx-1 dependent. Treatment with the S-protein-induced PGE 2 secretion was also ∼2.5-fold more effective than treatment with 229E (∗p ≤ 0.001, n = 4). Pretreatment with Panx-1 blockers, Probenecid, or the Panx-1 mimetic peptide before treatment with the S Prot or 229E did not result in significantly elevated concentrations of ATP. In addition, pretreatment with Scram before treatment with S protein or 229E resulted in elevated concentrations of PGE 2 comparable with cells treated with S protein or 229E alone, respectively (∗p ≤ 0.001, n = 4). Each value corresponds to the mean ± SD (n = 4). (C) Determination of IL-1β secretion from primary human airway epithelial cells for the control (C) and after S protein (S Prot) or hCoV-229E virus (229E) treatment in the presence or absence of Probenecid (Pro), Panx-1 mimetic peptide (pep), or the scrambled peptide (Sc). Relative to the control (C), SARS-CoV-2 S protein- and hCoV-229E-treatment-induced IL-1β secretion was also strongly Panx-1 dependent. Treatment with the S-protein-induced IL-1β secretion was similar to treatment with 229E (∗p ≤ 0.001, n = 4). Pretreatment with Panx-1 blockers, Probenecid, or the Panx-1 mimetic peptide before treatment with the S Prot or 229E did not result in significantly elevated concentrations of ATP. In addition, pretreatment with Scram before treatment with S protein or 229E resulted in elevated concentrations of IL-1β comparable with cells treated with S protein or 229E alone, respectively (∗p ≤ 0.043, n = 3, relative to control conditions). Each value corresponds to the mean ± SD (n = 3). It should be noted that although IL-1β secretion is correlated with Panx-1 channel activity, it is an indirect measure of cellular activation.

Article Snippet: SARS-CoV-2 (GISAID, EPI_ISL_406862) and Human Coronavirus 229E (ATCC, VR-740) were propagated in Vero E6 cells.

Techniques: Virus, Control, Activity Assay, Activation Assay

Journal: iScience

Article Title: Pannexin-1 channel opening is critical for COVID-19 pathogenesis

doi: 10.1016/j.isci.2021.103478

Figure Lengend Snippet: Panx-1 channel opening induced by SARS-CoV-2 is ACE-2, endocytosis, and furin dependent In contrast, Panx-1 channel opening in response to hCoV-229E is endocytosis dependent but ACE-2 and furin independent. (A–F) Representative snapshots of the time course of Etd uptake in airway epithelial cells for the untreated control at 0, 15, and 30 min. Images shown for each time point are in duplicate with brightfield (left) and fluorescence (right) channels. (G–L) Representative snapshots of the time course of Etd uptake in airway epithelial cells after treatment with S protein (1μg/mL) or 229E (0.1 MOI) or its purified S protein (5 μg/mL) at 0, 15, and 30 min. (M–R) Representative snapshots of the time course of Etd uptake in airway epithelial cells after pretreatment with furin (0.86 μg/mL) and then treatment with S protein (1μg/mL) or 229E (0.1 MOI) at 0, 15, and 30 min. (S) Quantification of the time course of Etd uptake for the airway epithelial cells that were untreated (black square) or treated with S protein alone (red circle) or S protein pretreated with human recombinant ACE-2 protein (0.4 μg/mL, blue upward triangle), endocytotic inhibitors (NH 4 Cl, 50 mmol/L; bafilomycin-A1, 50 nmol/L: and chloroquine, 100 μg/mL, green downward triangle), or furin (0.86 μg/mL, pink leftward triangle). Furin pretreatment of S protein resulted in the most significant uptake of Etd even relative to treatment with S protein alone (p ≤ 0.00025, n = 3, between 4 and 24 min for S protein and 4 and 42 min for furin-treated S protein compared with control conditions. Each value corresponds to the mean ± SD (n = 3). (T) Quantification of the time course of Etd uptake for the airway epithelial cells that were untreated (black square) or treated with the hCoV-229E (229E) alone (red circle) or 229E pretreated with human recombinant ACE-2 protein (0.4 μg/mL, blue upward triangle), endocytotic inhibitors (NH 4 Cl, 50 mmol/L; bafilomycin-A1, 50 nmol/L: and chloroquine, 100 μg/mL) (green downward triangle), or furin (0.86 μg/mL, pink leftward triangle). For this experiment, Panx-1 channel opening induced by hCoV-229E was only dependent on endocytosis but independent of ACE-2 and furin pathways (p ≤ 0.0012, n = 3) compared with control conditions. Denote the different scale between graphs S and T (2 folds increase in SARS-CoV-2 compared to 229E). Each value corresponds to the mean ± SD (n = 3).

Article Snippet: SARS-CoV-2 (GISAID, EPI_ISL_406862) and Human Coronavirus 229E (ATCC, VR-740) were propagated in Vero E6 cells.

Techniques: Control, Fluorescence, Purification, Recombinant

Journal: iScience

Article Title: Pannexin-1 channel opening is critical for COVID-19 pathogenesis

doi: 10.1016/j.isci.2021.103478

Figure Lengend Snippet:

Article Snippet: SARS-CoV-2 (GISAID, EPI_ISL_406862) and Human Coronavirus 229E (ATCC, VR-740) were propagated in Vero E6 cells.

Techniques: Saline, Electron Microscopy, RNAscope, Multiplex Assay, Blocking Assay, Sequencing, Membrane, Software