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

Journal: Cell reports

Article Title: Systematic analysis of purified astrocytes after SCI unveils Zeb2os function during astrogliosis

doi: 10.1016/j.celrep.2021.108721

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse monoclonal anti-BrDU , Cell Signaling Technology , Cat# 50230; RRID: AB_2799369.

Techniques: Virus, shRNA, Plasmid Preparation, Purification, Recombinant, Multiplex Assay, Transfection, RNAscope, Negative Control, Sequencing, Software, Flow Cytometry, Fluorescence, Microscopy, Laser-Scanning Microscopy

Journal: iScience

Article Title: Patterning the embryonic pulmonary mesenchyme

doi: 10.1016/j.isci.2022.103838

Figure Lengend Snippet: Computationally identified mesenchymal clusters represent spatially distinct populations (A) Violin plots showing the expression of Hoxb6 or Ptn in each cluster. (B) Fluorescence in situ hybridization for Hoxb6 and Ptn in E 11.5 lungs. Scale bar shows 25 μm. (C) Violin plots showing the expression of Lef1 or Foxp1 in each cluster. (D) E 11.5 lungs immunostained for cluster 0 marker Lef1 or for cluster 1 marker Foxp1 and counterstained with Hoechst. Scale bars show 25 μm. (E) Quantifications of Lef1 and Foxp1 intensity profiles emanating from the epithelium (for Lef1) or from the mesothelium (for Foxp1). Schematics show lines and direction along which intensity profiles were measured. Mean and SD are plotted (n = 4). (F) Schematic depicting the sub-epithelial and sub-mesothelial compartments of the mesenchyme. (G) Heatmap showing the expression of genes specific to either mesenchymal compartment. Genes (rows) are clustered based on the dendrogram to the right. Cells (columns) are clustered based on the dendrogram above, and each column is color-coded according to the original cluster identity from Figure 1 B. (H) UMAP of mesenchymal and smooth muscle cells color-coded according to the sum of their expression of either sub-epithelial or sub-mesothelial mesenchyme marker genes. Dotted line indicates the location of smooth muscle cells

Article Snippet: Rabbit polyclonal Foxp1 antibody , Cell Signaling , Cat# 2005; RRID: AB_2106979.

Techniques: Expressing, Fluorescence, In Situ Hybridization, Marker

Journal: iScience

Article Title: Patterning the embryonic pulmonary mesenchyme

doi: 10.1016/j.isci.2022.103838

Figure Lengend Snippet: Wnt signaling regulates cell identity in the embryonic pulmonary mesenchyme. (A) Bubble plot showing the enrichment percentage and adjusted p value of relevant GO terms identified based on genes upregulated in each mesenchymal cluster. (B) Heatmap showing the expression of Wnt-associated genes upregulated in either mesenchymal cluster. Activators and targets are colored in blue, inhibitors are colored in red. (C) Heatmap showing the expression of Wnt ligands, secreted inhibitors, and receptors detected in either mesenchymal cluster and in clusters containing cells from the mesothelium (meso), vascular endothelium (ve), epithelium (ep), and smooth muscle (sm). (D–G) Confocal sections and quantification of Lef1 and Foxp1 intensity profiles around branch L.L2 in lungs isolated at E 11.5 from CD1 embryos and immunostained for Lef1 or Foxp1 after treatment with either DMSO, LiCl (10 mM), or IWR1 (100 μM) for 24 h (n = 2–6). Yellow dashed lines indicate the border of the epithelium. Schematics show lines and direction along which intensity profiles were measured. Mean and SEM are plotted, and curves were compared using two-way ANOVA. (H–M) E 12.5 control and Tbx4-rtTA ; tet-O-Cre ; Ctnnb1 fl/fl lungs immunostained for Lef1 or Foxp1 and quantification of Lef1 and Foxp1 intensity profiles (n = 3). Low-magnification z-projections (H and I) and high-magnification confocal slices (J and K) are shown. Scale bars show 50 μm. ∗ indicates p<0.05, ∗∗ indicates p<0.001, and ∗∗∗ indicates p<0.0001

Article Snippet: Rabbit polyclonal Foxp1 antibody , Cell Signaling , Cat# 2005; RRID: AB_2106979.

Techniques: Expressing, Isolation, Control

Journal: iScience

Article Title: Patterning the embryonic pulmonary mesenchyme

doi: 10.1016/j.isci.2022.103838

Figure Lengend Snippet: Regulators and features of smooth muscle differentiation (A) Sections of E 12.5 Dermo1-Cre/+; Yap fl/fl ; mTmG/+ lungs and littermate controls immunostained for GFP and either Yap1, Lef1, Foxp1, or αSMA. Insets show zoomed-in view of the mesenchyme to highlight the decrease in mesenchymal Yap1 levels in mutants. Yap1 + cells in the mesenchyme of mutants are vascular endothelial cells (ve, indicated by white arrowheads), which are not targeted by Dermo1-Cre . ep is epithelium. Scale bars show 50 μm. (B) Scaled expression of genes involved in cytoskeleton, cell adhesion, and extracellular matrix versus cell loadings along DC1 compared to the expression profiles of the smooth muscle markers Acta2 and Myocd (dotted lines). Pearson correlation coefficients and significance are indicated and lines represent smoothed data with SE shaded in gray. (C) Simplified pathway diagram depicting the steps of proliferative metabolism and showing relevant enzymes at each step. Enzymes that are significantly downregulated along DC1 are indicated in bold red font, with a significance of spline fit indicated by asterisks. ∗ indicates p < 0.05, ∗∗ indicates p < 0.001, and ∗∗∗ indicates p < 0.0001

Article Snippet: Rabbit polyclonal Foxp1 antibody , Cell Signaling , Cat# 2005; RRID: AB_2106979.

Techniques: Expressing

Journal: iScience

Article Title: Patterning the embryonic pulmonary mesenchyme

doi: 10.1016/j.isci.2022.103838

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal Foxp1 antibody , Cell Signaling , Cat# 2005; RRID: AB_2106979.

Techniques: Recombinant, RNAscope, Multiplex Assay, Mutagenesis, Software, Sequencing

Journal: Cell

Article Title: Food Perception Primes Hepatic ER Homeostasis via Melanocortin-Dependent Control of mTOR Activation

doi: 10.1016/j.cell.2018.10.015

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: p-p70 S6 kinase antibody , Cell Signaling , Cat# 9234; RRID: AB_2269803.

Techniques: Virus, Plasmid Preparation, Recombinant, Western Blot, Electron Microscopy, Clinical Proteomics, Modification, Enzyme-linked Immunosorbent Assay, Reverse Transcription, BIA-KA, RNAscope, Multiplex Assay, Fluorescence, Sample Prep, Software

Journal: Developmental cell

Article Title: Specialized endothelial tip cells guide neuroretina vascularization and blood-retina-barrier formation

doi: 10.1016/j.devcel.2021.06.021

Figure Lengend Snippet: (A) Experimental strategy to delete Smad2 and Smad3 in ECs. Red arrows indicate TAM injections. (B and C) Western blot for SMAD2 or SMAD3 and β-actin using ECs isolated from lungs of WT ( Smad2l/l or Smad3l/l ) and Smad2iEKO and Smad3iEKO mice, respectively. (D) IsoB4/α-SMA double staining of P12 retinas of the indicated genotypes. (E) Quantification of vascular parameters of Smad2/3l/l and Smad2/3iEKO P12 retinas (mean ± SEM, each dot represents one retina, *p < 0.05, **p < 0.01, Mann-Whitney U test). (F) Experimental strategy to delete Smad4 in ECs. Red arrows indicate TAM injections. (G) IsoB4 and α-SMA double staining of P12 Smad4l/l and Smad4iEKO retinas. (H) Quantification of vascular parameters of P12 retinas of the indicated genotypes (mean ± SEM, each dot represents one retina, *p < 0.05, **p < 0.01, **p < 0.001, Mann-Whitney U test). (I) Double immunostainings for IsoB4 and APOE, VWF, TXNDR1, or PLVAP of retina flat mounts of the indicated genotypes at P12. A, artery; V, vein.

Article Snippet: Rabbit Anti-Smad3 (C67H9) monoclonal antibody , Cell Signaling , Cat#9523; RRID: AB_2193182.

Techniques: Western Blot, Isolation, Double Staining, MANN-WHITNEY

Journal: Developmental cell

Article Title: Specialized endothelial tip cells guide neuroretina vascularization and blood-retina-barrier formation

doi: 10.1016/j.devcel.2021.06.021

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit Anti-Smad3 (C67H9) monoclonal antibody , Cell Signaling , Cat#9523; RRID: AB_2193182.

Techniques: Recombinant, Saline, Fluorescence, Plasmid Preparation, Modification, Western Blot, cDNA Synthesis, SYBR Green Assay, Imaging, RNAscope, Multiplex Assay, Gene Expression, Software, Laser-Scanning Microscopy