Journal: Analytical and Bioanalytical Chemistry

Article Title: Emerging point-of-care biosensors for rapid diagnosis of COVID-19: current progress, challenges, and future prospects

doi: 10.1007/s00216-021-03377-6

Figure Lengend Snippet: Major diagnostic markers for COVID-19. Following the infection, the number of SARS-CoV-2 RNA increases dramatically at the early stage of infections, followed by an increase in the level of antibodies. Adapted with permission from

Article Snippet: , GENLISA™ Anti-SARS-Cov-2 (Covid-19) IgM Rapid Test , Paper-based biosensor , Krishgen Biosystems, India , Serum, plasma, whole blood , 15 min , 134.64 (per kit) , 97% , 97% , [ ] .

Techniques: Diagnostic Assay, Infection

Journal: Analytical and Bioanalytical Chemistry

Article Title: Emerging point-of-care biosensors for rapid diagnosis of COVID-19: current progress, challenges, and future prospects

doi: 10.1007/s00216-021-03377-6

Figure Lengend Snippet: Chip-based biosensors for potential antigen and antibody detection for COVID-19. a Miniaturized and high-throughput ELISA on a chip-based biosensor. The fingerstick blood is directly applied to an assay chamber. The slide is prepared by printing “stable” spots of capture antibodies (cAb) and “soluble” spots of the fluorescently tagged detection antibodies (dAb) on the polymer brush (1). dAb dissolves and binds to the analyte (2), the complexes diffuse and bind to the respective cAb spots (3), and subsequently (4) generates fluorescent signals. A mobile phone-based reader is coupled with the biosensor to provide quantitative readout. Adapted with permission from . b Multiplexed detection of IgG, IgM, and the viral antigen for SARS-CoV-2 based on fluorescence immunoassay using a centrifugal chip-based biosensor (FMS: fluorescent microsphere). Adapted with permission from . c A low-consumption electrochemical biosensor for antigen detection, which is powered directly by a mobile phone. Adapted with permission from

Article Snippet: , GENLISA™ Anti-SARS-Cov-2 (Covid-19) IgM Rapid Test , Paper-based biosensor , Krishgen Biosystems, India , Serum, plasma, whole blood , 15 min , 134.64 (per kit) , 97% , 97% , [ ] .

Techniques: High Throughput Screening Assay, Enzyme-linked Immunosorbent Assay, Fluorescence

Journal: Analytical and Bioanalytical Chemistry

Article Title: Emerging point-of-care biosensors for rapid diagnosis of COVID-19: current progress, challenges, and future prospects

doi: 10.1007/s00216-021-03377-6

Figure Lengend Snippet: Paper-based biosensors for potential antigen and antibody detection for COVID-19. a Monoplexed and multiplexed paper-based biosensors or lateral flow test strips for the detection of antibodies specific to the viruses. A customized phone application records the results along with the patient data for disease management. Adapted with permission from . b A paper-based biosensor which detects IgG and IgM for COVID-19. Adapted with permission from . c 3D-μPAD and its simple operation for simultaneous detection of three different antibodies for medical diagnosis. Adapted with permission from

Article Snippet: , GENLISA™ Anti-SARS-Cov-2 (Covid-19) IgM Rapid Test , Paper-based biosensor , Krishgen Biosystems, India , Serum, plasma, whole blood , 15 min , 134.64 (per kit) , 97% , 97% , [ ] .

Techniques:

Journal: Analytical and Bioanalytical Chemistry

Article Title: Emerging point-of-care biosensors for rapid diagnosis of COVID-19: current progress, challenges, and future prospects

doi: 10.1007/s00216-021-03377-6

Figure Lengend Snippet: Other point-of-care biosensors for potential antigen and antibody detection for COVID-19. a A nanoparticle-based portable bioluminescent immunosensor (ABS). Adapted with permission from . b An array of silicon nanowire biosensor for sensitive detection of disease biomarkers. Adapted with permission from . c A black phosphorus-based biosensor for electrochemical detection of IgG. Adapted with permission from . d A polysiloxane-modified thread-based biosensor for sensitive detection of pathogens. Adapted with permission from (196)

Article Snippet: , GENLISA™ Anti-SARS-Cov-2 (Covid-19) IgM Rapid Test , Paper-based biosensor , Krishgen Biosystems, India , Serum, plasma, whole blood , 15 min , 134.64 (per kit) , 97% , 97% , [ ] .

Techniques: Modification

Journal: Analytical and Bioanalytical Chemistry

Article Title: Emerging point-of-care biosensors for rapid diagnosis of COVID-19: current progress, challenges, and future prospects

doi: 10.1007/s00216-021-03377-6

Figure Lengend Snippet: Chip-based biosensors for potential nucleic acid detection for COVID-19. a A PDMS/paper hybrid chip-based biosensor for pathogen detection. Adapted with permission from . b A chip-based biosensor integrated with loop-mediated isothermal amplification (LAMP) and portable battery-powered heater. Adapted with permission from . c A lab-on-a-chip platform which consists of an array of ISFET chip-based biosensors coupled with a thermal controller. Adapted with permission from

Article Snippet: , GENLISA™ Anti-SARS-Cov-2 (Covid-19) IgM Rapid Test , Paper-based biosensor , Krishgen Biosystems, India , Serum, plasma, whole blood , 15 min , 134.64 (per kit) , 97% , 97% , [ ] .

Techniques: Amplification, Lab-on-a-Chip

Journal: Analytical and Bioanalytical Chemistry

Article Title: Emerging point-of-care biosensors for rapid diagnosis of COVID-19: current progress, challenges, and future prospects

doi: 10.1007/s00216-021-03377-6

Figure Lengend Snippet: Paper-based biosensors for potential nucleic acid detection for COVID-19. a A paper-based biosensor or lateral flow test strip for visualization of COVID-19 reverse transcription loop-mediated isothermal amplification (RT-LAMP) products. Adapted with permission from . b An integrated paper-based sample-to-answer biosensor for the detection of pathogens. Adapted with permission from . c A disposable and integrated paper-based biosensor for nucleic acid extraction, amplification, and detection. Adapted with permission from (197)

Article Snippet: , GENLISA™ Anti-SARS-Cov-2 (Covid-19) IgM Rapid Test , Paper-based biosensor , Krishgen Biosystems, India , Serum, plasma, whole blood , 15 min , 134.64 (per kit) , 97% , 97% , [ ] .

Techniques: Stripping Membranes, Amplification

Journal: Analytical and Bioanalytical Chemistry

Article Title: Emerging point-of-care biosensors for rapid diagnosis of COVID-19: current progress, challenges, and future prospects

doi: 10.1007/s00216-021-03377-6

Figure Lengend Snippet: Other point-of-care biosensors for potential nucleic acid detection for COVID-19. a An optomagnetic biosensor for real-time nucleic acid detection of SARS-CoV-2. Adapted with permission from . b Schematic diagram of an upconverting nanoparticle-based biosensor for pathogen detection. Adapted with permission from . c A cotton thread-based biosensor for nucleic acid detection for disease diagnosis. Adapted with permission from

Article Snippet: , GENLISA™ Anti-SARS-Cov-2 (Covid-19) IgM Rapid Test , Paper-based biosensor , Krishgen Biosystems, India , Serum, plasma, whole blood , 15 min , 134.64 (per kit) , 97% , 97% , [ ] .

Techniques:

Journal: Analytical and Bioanalytical Chemistry

Article Title: Emerging point-of-care biosensors for rapid diagnosis of COVID-19: current progress, challenges, and future prospects

doi: 10.1007/s00216-021-03377-6

Figure Lengend Snippet: Commercially available biosensors for COVID-19

Article Snippet: , GENLISA™ Anti-SARS-Cov-2 (Covid-19) IgM Rapid Test , Paper-based biosensor , Krishgen Biosystems, India , Serum, plasma, whole blood , 15 min , 134.64 (per kit) , 97% , 97% , [ ] .

Techniques: Biomarker Assay, Amplification

Journal: Journal of Medicinal Chemistry

Article Title: Non-Catalytic Inhibitors of the p38/MK2 Interface: Repurposing Approved Drugs to Target Neuroinflammation in Alzheimer’s Disease

doi: 10.1021/acs.jmedchem.5c01425

Figure Lengend Snippet: Structure-guided mapping and peptide validation of the p38/MK2 interaction interface. (A) The cocrystal structure of the p38/MK2 complex (PDB ID: 6TCA ). The molecular surface of p38 is shown in gray. The p38 docking groove is highlighted in yellow. MK2 is shown as green ribbons. (B) The MK2 D345-H400 docking motif bound to the p38 docking groove is colored based on its fragments tested in this study: D345-H400 is colored in green, I370–L393 in blue, and I370-L382 in red. (C) The binding curve from a fluorescence polarization assay showing high-affinity binding of FITC-labeled MK2 370–393 peptide to His-tagged p38 (EC 50 = 26.9 nM). (D) Dose–response curves from TR-FRET inhibition assays demonstrating that both MK2 370–393 and 369–382 peptides disrupt the p38/MK2 complex (IC 50 = 0.42 μM and 4.26 μM, respectively).

Article Snippet: Thirty min incubation in 5% nonfat dry milk (BioRad, catalog no. 170–6404) in TBST buffer (20 mM Tris-base, 150 mM NaCl, and 0.05% Tween 20) at room temperature was used to block membranes. p38α MAPK mouse mAb (Cell Signaling Technology, catalog no. 9217) was used to blot the membrane at 4 °C overnight.

Techniques: Biomarker Discovery, Binding Assay, Fluorescence, Labeling, Inhibition

Journal: Journal of Medicinal Chemistry

Article Title: Non-Catalytic Inhibitors of the p38/MK2 Interface: Repurposing Approved Drugs to Target Neuroinflammation in Alzheimer’s Disease

doi: 10.1021/acs.jmedchem.5c01425

Figure Lengend Snippet: Virtual screening and molecular dynamics analysis identify nilotinib as a candidate p38/MK2 PPI inhibitor. (A) Distribution of MM-GBSA binding free energies (Δ G bind ) from virtual screening of 1,040 FDA-approved drugs docked to the p38 docking groove. The p38 crystal structure (PDB ID: 6TCA ) was used for the modeling studies. Compounds with Δ G bind values more than two standard deviations below the mean (red bars, <−60.9 kcal/mol) were prioritized for further analysis. (B) Representative binding pose of carvedilol highlighting key interactions with the p38 docking groove, including hydrogen bonds with Val158, Glu160, and His126 (yellow lines), hydrophobic interactions with the nonpolar pocket defined by Ile116, Leu122, Leu130, and Val158, and a pi-pi stacking with His126 (cyan line). (C) Carvedilol’s carbazole moiety binds within the hydrophobic cleft of the p38 docking groove, which is shown as a molecular surface representation colored by electrostatic potential (red = negative, blue = positive). (D) Root-mean-square deviation (RMSD) plots from three 200 ns molecular dynamics simulations of the p38–nilotinib complex. The RMSD of protein backbone atoms is shown in aquamarine, and nilotinib in red. The PDB IDs of the p38 structures used for the modeling are indicated in the lower-left corners. (E) The representative binding pose of nilotinib obtained after 200 ns MD simulation (a final snapshot of one of the MDs), highlighting pi-pi and H-bond interactions with His126, the H-bonding with Glu160, and multiple water-bridged contacts that stabilize ligand orientation within the groove. (F) Structural overlay of the nilotinib–p38 complex with the p38/MK2 cocrystal structure, illustrating displacement of key MK2 anchoring residues Ile372 and Ile375 by nilotinib. P38 is shown as green ribbons, the p38 docking groove as gray molecular surface, and MK2 as red ribbons.

Article Snippet: Thirty min incubation in 5% nonfat dry milk (BioRad, catalog no. 170–6404) in TBST buffer (20 mM Tris-base, 150 mM NaCl, and 0.05% Tween 20) at room temperature was used to block membranes. p38α MAPK mouse mAb (Cell Signaling Technology, catalog no. 9217) was used to blot the membrane at 4 °C overnight.

Techniques: Binding Assay

Journal: Journal of Medicinal Chemistry

Article Title: Non-Catalytic Inhibitors of the p38/MK2 Interface: Repurposing Approved Drugs to Target Neuroinflammation in Alzheimer’s Disease

doi: 10.1021/acs.jmedchem.5c01425

Figure Lengend Snippet: Validation of nilotinib as a p38/MK2 PPI Inhibitor. (A) Thermal shift assay (TSA) showing dose-dependent stabilization of recombinant His-tagged p38 by nilotinib (Δ T max = 8.22 °C), consistent with direct binding. (B) TSA profile for SR318, a type II ATP-competitive p38 inhibitor, used as a positive control (Δ T max = 13.47 °C). (C) Nilotinib competes with His-MK2 346–400 fragment for VF-p38 in a cell lysate-based TR-FRET assay. (D) Quantitative qRT-PCR analysis showing that nilotinib significantly ( p -value <0.05) suppresses LPS-induced TNF-α, IL-6, and IL-1β expression in HMC3 microglial cells. P38 inhibitors SR318 and VX-745 were used as positive controls. (E) Nilotinib disrupts the endogenous p38/MK2 complex in HMC3 cells, as shown by coimmunoprecipitation, correlating with cytokine suppression. (F) qRT-PCR analysis showing that nilotinib suppresses LPS/IFNγ-induced TNF-α expression in the human iPSC-derived microglia (iMGL). (G) TR-FRET assay with recombinant p38 and MK2 proteins purified from E. coli demonstrated direct inhibition of the complex by nilotinib (IC 50 = 2.2 μM). In contrast, ATP-site inhibitors VX-745 and SR318 failed to disrupt the interaction, supporting a non-ATP-competitive mechanism for nilotinib activity. (H) Nilotinib demonstrates a weak inhibition of p38/ATF2 PPI (IC 50 > 30 μM, maximal inhibition ∼ 37%) in a TR-FRET assay with recombinant purified His-p38 and GST-ATF2. The inhibition of His-p38/GST-MK2 PPI by nilotinib was monitored in parallel.

Article Snippet: Thirty min incubation in 5% nonfat dry milk (BioRad, catalog no. 170–6404) in TBST buffer (20 mM Tris-base, 150 mM NaCl, and 0.05% Tween 20) at room temperature was used to block membranes. p38α MAPK mouse mAb (Cell Signaling Technology, catalog no. 9217) was used to blot the membrane at 4 °C overnight.

Techniques: Biomarker Discovery, Thermal Shift Assay, Recombinant, Binding Assay, Positive Control, Quantitative RT-PCR, Expressing, Derivative Assay, Purification, Inhibition, Activity Assay

Journal: Journal of Medicinal Chemistry

Article Title: Non-Catalytic Inhibitors of the p38/MK2 Interface: Repurposing Approved Drugs to Target Neuroinflammation in Alzheimer’s Disease

doi: 10.1021/acs.jmedchem.5c01425

Figure Lengend Snippet: Chemical structures of nilotinib and ten analogs evaluated for p38/MK2 PPI inhibition using a TR-FRET assay with recombinant purified proteins. IC 50 values are shown for compounds exhibiting measurable activity; compounds with less than 50% inhibition at 30 μM are indicated as not determined (N.D.).

Article Snippet: Thirty min incubation in 5% nonfat dry milk (BioRad, catalog no. 170–6404) in TBST buffer (20 mM Tris-base, 150 mM NaCl, and 0.05% Tween 20) at room temperature was used to block membranes. p38α MAPK mouse mAb (Cell Signaling Technology, catalog no. 9217) was used to blot the membrane at 4 °C overnight.

Techniques: Inhibition, Recombinant, Purification, Activity Assay

Journal: Journal of Medicinal Chemistry

Article Title: Non-Catalytic Inhibitors of the p38/MK2 Interface: Repurposing Approved Drugs to Target Neuroinflammation in Alzheimer’s Disease

doi: 10.1021/acs.jmedchem.5c01425

Figure Lengend Snippet: Field-based QSAR maps illustrating physicochemical features of nilotinib analogs associated with p38/MK2 PPI inhibition. (A) Compounds 1–6 (white) and 7–10 (orange) docked into the p38 binding groove. The molecular surface of the binding groove is colored based on the electrostatic potential, ranging from the most positive (blue) to the most negative (red) charge. (B) Steric field map showing regions where steric bulk is favorable (green). The pyridine–pyrimidine system is positioned within favorable steric zones, supporting its critical role in activity. (C) Hydrophobic field map with yellow-green and gray surfaces representing positive and negative hydrophobic contributions, respectively. (D) Electrostatic field map colored by potential (red - negative, blue - positive). (E) Hydrogen bond acceptor field map. Red contours indicate favorable contributions of H-bond acceptors, while the magenta contour indicates unfavorable contributions of H-bond acceptors. (F) Hydrogen bond donor field map. The blue-violet contour indicates the region favorable for H-bond donors. The cyan field map indicates the area unfavorable for the H-bond donors.

Article Snippet: Thirty min incubation in 5% nonfat dry milk (BioRad, catalog no. 170–6404) in TBST buffer (20 mM Tris-base, 150 mM NaCl, and 0.05% Tween 20) at room temperature was used to block membranes. p38α MAPK mouse mAb (Cell Signaling Technology, catalog no. 9217) was used to blot the membrane at 4 °C overnight.

Techniques: Inhibition, Binding Assay, Activity Assay

Journal: Journal of Medicinal Chemistry

Article Title: Non-Catalytic Inhibitors of the p38/MK2 Interface: Repurposing Approved Drugs to Target Neuroinflammation in Alzheimer’s Disease

doi: 10.1021/acs.jmedchem.5c01425

Figure Lengend Snippet: Development of a lysate-based TR-FRET platform for high-throughput screening of p38/MK2 PPI inhibitors. (A) The preferential binding of MK2 to p38α and p38β isoforms was determined by Flag-immunoprecipitation in HEK293T cells. (B) Isoform selectivity of MK2 binding was validated by TR-FRET using lysates coexpressing GST-tagged MK2 and Venus-Flag (VF)-tagged p38 isoforms. Robust signal was observed for p38α and p38β, with negligible interaction detected for p38γ and p38δ. (C) TR-FRET assay shows stable signal over 48 h postantibody addition, indicating excellent temporal stability. (D) The platform tolerates up to 10% DMSO without signal degradation, supporting its suitability for screening applications. (E) Pilot screen of 2036 compounds from the Emory Enriched Library (EEL) in 1536-well format identified 48 compounds that inhibited the p38/MK2 interaction by ≥ 50% relative to vehicle control. Gray dots indicate fluorescence assay-interfering compounds.

Article Snippet: Thirty min incubation in 5% nonfat dry milk (BioRad, catalog no. 170–6404) in TBST buffer (20 mM Tris-base, 150 mM NaCl, and 0.05% Tween 20) at room temperature was used to block membranes. p38α MAPK mouse mAb (Cell Signaling Technology, catalog no. 9217) was used to blot the membrane at 4 °C overnight.

Techniques: High Throughput Screening Assay, Binding Assay, Immunoprecipitation, Control, Fluorescence

Journal: Journal of Medicinal Chemistry

Article Title: Non-Catalytic Inhibitors of the p38/MK2 Interface: Repurposing Approved Drugs to Target Neuroinflammation in Alzheimer’s Disease

doi: 10.1021/acs.jmedchem.5c01425

Figure Lengend Snippet: α 1 -Adrenergic antagonists disrupt the p38/MK2 interface and suppress cytokine production in microglial cells. (A) Chemical structures of doxazosin, terazosin, and alfuzosin, three α 1 -adrenergic receptor antagonists identified from the high-throughput screen. (B) Dose–response TR-FRET assays using recombinant purified p38 and MK2 proteins demonstrate that all three compounds inhibit the p38/MK2 protein–protein interaction, with IC 50 values of 4.4 μM (doxazosin), 6.2 μM (terazosin), and 6.9 μM (alfuzosin). (C) The compound activity was confirmed in a cell lysate-based TR-FRET format, showing a moderate reduction in potency relative to the recombinant protein assay. (D) In a complementary TR-FRET assay using HEK293T lysates coexpressing VF-tagged p8 and a His-tagged MK2 346–400 docking peptide, all three α 1 -antagonists and nilotinib dose-dependently disrupted peptide binding to p38, consistent with direct competition at the docking interface. (E) qRT-PCR analysis in HMC3 microglial cells shows that all three compounds significantly ( p -values <0.05) suppressed LPS-induced expression of TNF-α, IL-6, and IL-1β, similarly to known p38 inhibitors SR318 and VX745, demonstrating effective functional inhibition of p38/MK2 signaling in a disease-relevant context.

Article Snippet: Thirty min incubation in 5% nonfat dry milk (BioRad, catalog no. 170–6404) in TBST buffer (20 mM Tris-base, 150 mM NaCl, and 0.05% Tween 20) at room temperature was used to block membranes. p38α MAPK mouse mAb (Cell Signaling Technology, catalog no. 9217) was used to blot the membrane at 4 °C overnight.

Techniques: High Throughput Screening Assay, Recombinant, Purification, Activity Assay, Binding Assay, Quantitative RT-PCR, Expressing, Functional Assay, Inhibition

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: TL1A is an epithelial cytokine expressed in alveolar epithelium and airway basal cells in human healthy and asthmatic lungs. (A) Single-cell RNA-seq analysis of TNFSF15 ( TL1A ) expression in the LungMAP single-cell human lung atlas. Uniform manifold projection (UMAP) plots show the clustering of 347,970 lung cells (10 single-cell datasets, 148 normal human lung samples from 104 donors: adult, child, and adolescent). Results are visualized using ShinyCell and are based upon data generated by the LungMAP Consortium and downloaded from http://www.lungmap.net . (B and C) Single-cell RNA-seq analysis of TNFSF15 ( TL1A ) expression in epithelial cells from human healthy (B) and asthmatic (C) lungs. t-SNE plots show clustering of 26,154 epithelial cells in upper and lower airways and lung parenchyma in healthy lungs (B; 17 human samples: 6 alveoli and parenchyma, 9 bronchi, 2 nasal), and 25,146 epithelial cells from lower airways in healthy and asthmatic lungs (C; 12 human samples: 15,033 cells from 6 asthma bronchi; 10,113 cells from 6 control bronchi). t-SNE plots were extracted from data obtained by the human lung single-cell atlas and downloaded from https://asthma.cellgeni.sanger.ac.uk .

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: RNA Sequencing, Expressing, Generated, Control

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: Single-cell RNA-seq analysis of IL33 and TSLP expression in human lungs and gating strategy for analysis of mouse lung epithelial cells by flow cytometry. (A and B) Single-cell RNA-seq analysis of IL33 and TSLP expression in epithelial cells from human healthy (A) and asthmatic (B) lungs. t-SNE plots show clustering of 26,154 epithelial cells in upper and lower airways and lung parenchyma in healthy lungs (A; 17 human samples: 6 alveoli and parenchyma, 9 bronchi, 2 nasal), and 25,146 epithelial cells from lower airways in healthy and asthmatic lungs (B; 12 human samples: 15,033 cells from 6 asthma bronchi; 10,113 cells from 6 control bronchi). t-SNE plots were extracted from data obtained by the human lung single-cell atlas , and downloaded from https://asthma.cellgeni.sanger.ac.uk . (C) Gating strategy of Epcam + epithelial cells and CD31 + endothelial cells in the lung of a naïve WT mouse. (D and E) Immunohistofluorescence staining of lung tissue sections (naïve wild type C57BL/6J mouse, steady state) with two distinct rat IgG1 isotype controls (rat IgG1 clone eBRG1, D, red; rat IgG1 clone RB40.34, E, red) for the anti-TL1A antibody (rat IgG1, MAB7441, clone 293327). Double staining was performed with antibodies against RAGE (D, green) or IL-33 (E, green). Images are representative of two independent experiments. Scale bar, 10 μm.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: RNA Sequencing, Expressing, Flow Cytometry, Control, Immunohistofluorescence, Staining, Double Staining

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: TL1A is expressed in mouse alveolar epithelium at steady state. (A) Visualization of Tnfsf15 (TL1A) expressing cells in the LungMAP single-cell mouse lung atlas. UMAP plots show the clustering of 95,658 lung cells (17 samples from late developmental stage to postnatal day 28). The different cell types in the lungs of naïve mice are indicated on the left. Results are visualized using ShinyCell and are based upon data generated by the LungMAP Consortium and downloaded from http://www.lungmap.net . (B) Single-cell RNA-seq analysis of Tnfsf15/TL1A and Il33 gene expression in mouse lung epithelium. UMAP plots show clustering and cell type annotation of 12,536 mouse lung epithelial cells (seven samples from the emergence of the alveolus to postnatal day 28) . The number and percentage of epithelial cells expressing Tnfsf15/TL1A , Il33 , or both are indicated on the right. Results are visualized using ShinyCell and are based upon data obtained by and downloaded from http://www.lungmap.net . (C) Flow cytometry analysis of cell surface TL1A expression on live CD31 + CD45 − endothelial cells and Epcam + CD31 − CD45 − epithelial cells in the lung of a naïve wild type C57BL/6J mouse at steady state. (D and E) Immunohistofluorescence staining of lung tissue sections (naïve wild type C57BL/6J mouse, steady state) with antibodies against TL1A (D and E) and RAGE (D) or IL-33 (E) proteins. A tyramide signal amplification (TSA)-based immunofluorescence method was used to detect TL1A-expressing cells in situ. Images are representative of two independent experiments. Scale bar, 10 μm.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: Expressing, Generated, RNA Sequencing, Gene Expression, Flow Cytometry, Immunohistofluorescence, Staining, Amplification, Immunofluorescence, In Situ

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: High throughput proteomic analyses of lung ILC2s stimulated ex vivo with IL-33 and/or TL1A. (A) Flow cytometry of cultured lung ILC2s ex vivo. Representative histograms of ST2, CD90.2, Sca-1, CD25, ICOS, KLRG1, and DR3 expression at the surface of cultured ILC2s, 3 days after ILC2 cell isolation from the lung and ex vivo culture in the presence of IL-2. Phenotypic analysis was performed on live Lin – CD45 + cells. (B–D) Large-scale label-free proteomic analyses of mouse lung ILC2s after ex vivo overnight stimulation with rIL-2 ± rIL-33 ± rTL1A. Volcano plots of IL-33-stimulated ILC2s (B) or TL1A-stimulated ILC2s (C) compared with non-stimulated cells (NS; in culture with IL-2 alone). Volcano plot of IL-33/TL1A-stimulated ILC2s compared to IL-33-stimulated cells (D). Statistical analysis of protein abundance values was performed from different biological replicate experiments ( n = 6 for NS and IL33 stimulation; n = 3 for TL1A and IL33/TL1A stimulations), using a Student’s t test (log 10 P value, vertical axis). Proteins found as significantly over or under-expressed (P < 0.05 and abs[log 2 fold change] >1) are shown in black. Representative examples of proteins found modulated in each comparison are shown in color. (E) Flow cytometry of cultured lung ILC2s after 14 h of co-stimulation with IL-33 and TL1A in the presence of IL-2 (ILC2 culture used in ). Intracellular cytokine staining revealed that >99% of ILC2s co-expressed IL-9 and IL-13 intracellularly. Phenotypic analysis was performed on live Lin − CD45 + CD90.2 + cells.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: High Throughput Screening Assay, Ex Vivo, Flow Cytometry, Cell Culture, Expressing, Cell Isolation, Quantitative Proteomics, Comparison, Staining

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: TL1A synergizes with IL-33 to induce an IL-9-producing ILC9 phenotype in lung ILC2s. (A and B) Large-scale label-free proteomic analyses of ILC2s isolated from pooled lungs of IL-33-treated Rag2 −/− C57BL/6 J mice and cultured with IL-2 prior to overnight stimulation with rIL-2 ± rIL-33 ± rTL1A. Volcano plot of IL-33/TL1A-stimulated ILC2s (ILC9 cells) compared with nonstimulated cells (NS; in culture with IL-2 alone) (A). Statistical analysis of protein abundance values was performed from different biological replicate experiments ( n = 6 for NS; n = 3 for IL33/TL1A stimulation) using a Student’s t test (log 10 P value, vertical axis). Proteins found as significantly over or under-expressed (P < 0.05 and abs[log 2 fold change] >1) are shown in black. Examples of proteins modulated in both IL-33/TL1A-stimulated ILC2s and IL-33-stimulated ILC2s are shown in blue. Proteins shown in red are representative of molecules specifically modulated in IL-33/TL1A-stimulated ILC2s (A). Heat-map of fold changes of selected proteins in three independent biological replicates (B). (C–K) Analysis of ILC2s isolated from pooled lungs of IL-33-treated Rag2 −/− C57BL/6 J mice , and cultured with IL-2 prior to 14 h stimulation with rIL-2 ± rIL-33 ± rTL1A. Flow cytometry analysis of live Lin − CD45 + cells (C, E, and J), frequency of IL-9 high ILC2s (percentage of live Lin − CD45 + CD90.2 + cells) (D and K), and MFI fold change of IL-9 in ILC2s (E), after cytokines treatment and restimulation by PMA, ionomycin, and brefeldin A (4 h, C–E) or brefeldin A (4 h, J and K). Concentration of IL-9 secreted by ILC2s, measured by ELISA (F). Relative STAT5 mRNA expression levels measured by real-time qPCR (G). Samples were normalized to the expression of HPRT and are shown relative to IL-2-stimulated ILC2s. Immunoblot analysis of activated phosphorylated STAT5 (pSTAT5) and α-tubulin (H) or β-actin (I); Arrowheads indicate the migration of the protein of interest; cropped images. Cultured ILC2s were treated with rIL-2 + rIL-33 + rTL1A and increasing doses of a STAT5 inhibitor (STA5i, CAS 285986-31-4) or control vehicle (DMSO) (I–K). Numbers inside outlined areas (C) indicate percent of cells in the relevant gate. Each symbol represents an individual biological replicate (D–G and K). Data are pooled from six (D and E), six to eight (F) or three (G and K) independent experiments, or are representative of six (C and E) or three (H–J) independent experiments. Data are expressed as mean (±SEM) with P values determined by one-way ANOVA followed by Tukey’s multiple-comparisons test (D–G and K): ns not significant, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Source data are available for this figure: .

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: Isolation, Cell Culture, Quantitative Proteomics, Flow Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Migration, Control

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: IL-33 and TL1A synergistically induce IL-9-producing ILC2s ex vivo. (A) Analysis of cultured lung ILC2s 14 h after ex vivo stimulation by rIL-2 (20 ng/ml) ± rIL-33 (20 ng/ml) ± rTL1A (50 ng/ml). Flow cytometry analysis of live Lin − CD45 + cells and frequency of IL-9 high ILC2s (percentage of live Lin − CD45 + CD90.2 + cells) after cytokine treatment and incubation with brefeldin A (4 h), without restimulation by PMA and ionomycin. Numbers inside outlined area indicate percent of cells in the relevant gate and data are representative of eight independent experiments. (B) Concentration of IL-9 secreted by ILC2s treated with rIL-2 (20 ng/ml) and various concentrations of rIL-33 and rTL1A measured by ELISA. (C and D) MFI of nuclear factor IRF4 (C) and flow cytometry (D) of ILC2s 14 h after ex vivo stimulation of cultured ILC2s by rIL-2 (20 ng/ml) ± rIL-33 (20 ng/ml) ± rTL1A (50 ng/ml). Numbers inside outlined areas (D) indicate percent of cells in the relevant gate and data are representative of three independent experiments. (E) Immunoblot analysis of JunB and α-tubulin14 h after cytokine stimulation of lung ILC2s; Arrowheads indicate the migration of the protein of interest; cropped image. Data are representative of three independent experiments. (F–H) Relative mRNA expression levels by real time qPCR, 14 h after cytokine stimulation of lung ILC2s. Samples were normalized to the expression of HPRT and data are expressed relative to IL-2-stimulated ILC2s (F) or relative to HPRT mRNA quantity (G and H). (I and J) Analysis of mouse lung ILC2s 14 h after ex vivo stimulation by rIL-33 + rTL1A ± rIL-2 ± rIL-7 ± rTSLP. Frequency of IL-9 high ILC2s (Lin − CD45 + CD90.2 + cells), after cytokines treatment and re-stimulation by PMA, ionomycin and brefeldin A (4 h, I). Concentration of IL-9 secreted by ILC2s, measured by ELISA (J). (K) Concentration of IL-9 (ELISA) secreted by ILC2s 14 h after ex vivo stimulation by rIL-2 ± rIL-33 ± rIL-4 ± rTGF-β. Each symbol represents an individual biological replicates with n = 2–5 independent experiments (A–C and F–K). Data are expressed as mean (±SEM) with P values determined by one-way ANOVA followed by Tukey’s (A, C, and F–J) or Dunnett’s (B and K) multiple-comparisons tests: ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. In H, all significant P values are annotated with stars, all other comparisons are not significant. Source data are available for this figure: .

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: Ex Vivo, Cell Culture, Flow Cytometry, Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Migration, Expressing

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: IL-33 and TL1A induce phenotypic changes in cultured lung ILC2s at the protein and mRNA levels. (A–J) Analysis of mouse lung ILC2s 14 h after ex vivo stimulation by rIL-2 ± rIL-33 ± rTL1A. MFI of the indicated cell surface markers determined by flow cytometry (A, B, D, and E). Relative mRNA expression levels of various genes (C and F–I), including genes characteristic of ILC1s or ILC3s (I), determined by real-time qPCR, 14 h after cytokine stimulation of lung ILC2s. Samples were normalized to the expression of HPRT and data are expressed as relative to HPRT mRNA quantity. Concentration of IL-5 or IL-13 in cell supernatants, measured by ELISA assay (J). Each symbol represents an individual biological replicate from independent experiments (A–J). Data are expressed as mean (±SEM) with P values determined by unpaired two-tailed Student’s t test (B, E, and J) or one-way ANOVA followed by Tukey’s multiple-comparisons test (A, C, D, and F–I): ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001. In I, all significant P values are annotated with stars, all other comparisons are not significant.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: Cell Culture, Ex Vivo, Flow Cytometry, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: TL1A cooperates with IL-33 for induction of IL-9 high ILC2s in vivo. (A) Treatment schedule of naïve wild type (WT, C57BL/6J) mice. (B) Gating strategy of IL-9 high IL-5 + IL-13 + ILC2s. (C–I) Flow cytometry of IL-5 + IL-13 + ILC2s gated on live ILCs (Lin − CD45 + CD90.2 + cells) (C) and IL-9 high ILC2s gated on live IL-5 + IL-13 + ILC2s (E), frequency of lung IL-5 + IL-13 + ILC2s among live ILCs (D), IL-9 high ILC2s among live IL-5 + IL-13 + ILC2s (F), and IL-9 high IL-13 + ILC2s among live ILCs (G) or IL-9 high ILCs (H), and concentration of IL-9 in BAL fluids (ELISA assay, I) of WT mice 14 h after a single i.n. administration of PBS or rIL-33 (1 μg) and/or rTL1A (5 μg). Numbers inside outlined areas indicate the percent of cells in the relevant gate and data are representative of two independent experiments (C and E). Each symbol represents an individual mouse and data are pooled from two independent experiments. Data are expressed as mean (±SEM) with P values determined by one-way ANOVA followed by Tukey’s (D) or Dunnett’s (F, G, and I) multiple-comparisons tests: ns, not significant, ** P < 0.01, **** P < 0.0001. (J) Frequency of lung eosinophils (Gr1 low Siglec-F + CD11c − cells) among live CD45 + cells, at day 7 after a single i.n. exposure to rIL-33 or rIL-33 plus rTL1A. Each symbol represents an individual mouse and data are pooled from two independent experiments. Data are expressed as mean (±SEM) with P values determined by unpaired two-tailed Student’s t test: * P < 0.05. (K and L) Multiphoton imaging (K) and intravital microscopy (L) of whole lungs of INFER IL-9 fluorescent reporter mice, with detection of IL-9-eGFP + ILC2s (green) and staining of blood vessels (red) and collagen fibers (blue), 16–18 h after a single i.n. administration of IL-33/TL1A combination (1 μg rIL-33 plus 5 μg rTL1A). To increase the numbers of lung IL-9 high ILC2s accessible to in vivo imaging, the single i.n. exposure to IL-33/TL1A combination was performed after prior expansion of lung ILC2s by repeated i.p. injections of IL-33 (K and L). Multiphoton image (K) is a 3D reconstitution of stitched images (7 × 7 tiles and 181 z-stack). Time-lapse images (L) illustrate the migratory behavior of IL-9-eGFP + ILC2s. Time in h/min/s. Scale bars: K, 300 μm; L, 20 μm.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: In Vivo, Flow Cytometry, Concentration Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Imaging, Intravital Microscopy, Staining, In Vivo Imaging

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: IL-33 and TL1A synergistically induce IL-9-producing ILC2s in vivo. (A) Gating strategy and representative flow cytometry plots of live lung ILCs (live Lin − CD45 + CD90.2 + cells), live lung IL-5 + IL-13 + ILC2s (live IL-5 + IL-13 + ILCs) and live lung IL-9 high ILC2s (live IL-9 high IL-5 + IL-13 + ILC2s) in vivo in wild type (WT) C57BL/6J mouse, 14 h after a single i.n. administration of rIL-33 (1 μg) and rTL1A (5 μg). (B) Verification of the absence of contamination of the IL-5 + IL-13 + ILC2s and IL-9 high ILC2s populations by TCR + cells (T cells and NKT cells) using anti-TCRβ and anti-TCRγδ antibodies. (C) Confirmation of the expression of IL-5 and IL-13 in live Lin − CD3/TCR − NK1.1 − CD45 + CD90.2 + lung ILCs using antibodies against CD3/TCR and NK1.1 with a different fluorescence from the Lin cocktail (CD4, CD19, CD45R, CD11b, CD11c, Ter119, Ly6G, FcεRI). (D and E) Frequency of lung IL-9 high Lin − cells among live CD45 + cells (D), and flow cytometry of IL-9 high IL-13 + ILC2s (live IL-9 high IL-13 + Lin − CD45 + CD90.2 + cells) (E) of WT mice 14 h after a single i.n. administration of PBS or rIL-33 (1 μg) and/or rTL1A (5 μg). Numbers inside outlined areas indicate the percent of cells in the relevant gate. (F) Frequency of lung IL-9 high Lin − cells among live CD45 + cells of WT mice pretreated with six daily i.p. injections of rIL-33 (days 1–6) prior to one i.n. injection of PBS or rIL-33 and/or rTL1A (day 7). Flow cytometry analyses were performed on day 8. (G) Frequency of IL-9 high ILC2s among live ILCs (Lin − CD45 + CD90.2 + cells) in the lungs of WT mice 6 h after a single i.n. administration of A. alternata extract (12.5 μg), with (αIL-2 mAb) or without (Iso, isotype control mAb) IL-2 blockade. (H and I) Analysis of IL-9 and TL1A release in BAL fluids by ELISA at different time points after the third exposure to A. alternata in a chronic exposure model (repeated i.n. administration of 12.5 μg A. alternata at days 0, 3, and 6). Each symbol represents an individual mouse and data are pooled from two (D and G) or three (F, H, and I) independent experiments. Data are expressed as mean (±SEM) with P values determined by unpaired two-tailed Student’s t tests (G) or one-way ANOVA followed by Dunnett’s multiple-comparison test (D, F, H, and I): * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: In Vivo, Flow Cytometry, Expressing, Fluorescence, Injection, Control, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Comparison

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: Related to . Endogenous IL-9-producing ILC2s accumulate around blood vessels after IL33/TL1A treatment in vivo. IL9-eGFP + ILC2s (green), blood vessels (Evans Blue/red), and collagen fibers (second harmonic generation/blue) were visualized by multiphoton imaging in the cleared lung of INFER IL9 fluorescent reporter mice 16–18 h after administration of IL33/TL1A combination. 360° rotation of a 3D static representation at a frame rate of 25 fps (500 frames per 20 sec).

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: In Vivo, Imaging

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: Related to . Endogenous IL-9-producing ILC2s migrate along collagen fibers after IL33/TL1A treatment in vivo. IL9-eGFP + ILC2s (green), blood vessels (Evans Blue/red), and collagen fibers (second harmonic generation/blue) were visualized by lung intravital multiphoton imaging of INFER IL9 fluorescent reporter mice 16–18 h after administration of IL33/TL1A combination. Time in h/min/s. Playback speed: 600.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: In Vivo, Imaging

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: Endogenous TL1A functions as an epithelial alarmin rapidly released after allergen exposure. (A) Treatment schedule of naïve wild type (WT, C57BL/6J) mice. (B–F) Analysis of TL1A and IL-33 release in BAL fluids after a single allergen exposure. TL1A (B and E), IL-33 (C and F), and LDH (D) levels in BAL fluids were determined by ELISA (B, C, E, and F) or LDH (D) assays, 15 min (B–D) or at different time points (E and F) after a single i.n. administration of A. alternata extract (12.5 μg). Each symbol represents an individual mouse and data are pooled from two independent experiments (B–F). Data are expressed as mean (±SEM) with P values determined by one-way ANOVA followed by Tukey’s (B–D) or Dunnett’s (E and F) multiple-comparisons tests: ** P < 0.01, *** P < 0.001, **** P < 0.0001. (G–K) Analysis of TL1A release in cell supernatants after exposure of TL1A-expressing cells to A. alternata or bee venom phospholipase A2 (PLA2). U2OS epithelial cells transfected with a mouse TL1A-Flag expression vector (mTL1A-Flag vector) or control vector were analyzed by indirect immunofluorescence microscopy with anti-mTL1A and anti-Flag antibodies (G). Scale bar, 20 μm. TL1A (H and J) and LDH (I and K) levels in cell supernatants were determined by ELISA (H and J) or LDH cytotoxicity assays (I and K) 15 min after treatment with A. alternata extract ( A. alternata , H and I) or 1 h after treatment with bee venom PLA2 (J and K). NT, not treated. Each symbol represents an individual biological replicate and data are pooled from three independent experiments (H–K). Data are expressed as mean (±SEM) with P values determined by unpaired two-tailed Student’s t tests (treatment versus NT): ** P < 0.01, **** P < 0.0001.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Transfection, Plasmid Preparation, Control, Immunofluorescence, Microscopy, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: Endogenous TL1A is important for early induction of IL-9 high ILC2s after allergen exposure. (A) Treatment schedule of naïve WT mice. (B) IL-9 mRNA levels in the lungs analyzed by qPCR at different time points after a single allergen exposure. Data are expressed as relative to IL-9 mRNA levels in mice treated with PBS. (C–H) Flow cytometry and frequency of IL-9 high Lin − cells among live CD45 + cells (C and D) and IL-9 high ILC2s among live ILCs (Lin − CD45 + CD90.2 + cells) (E and F), flow cytometry (G), and MFI of IRF4 expression in ILC2s (H), in the lungs of WT mice 6 h after a single i.n. administration of A. alternata extract (12.5 μg), with (αTL1A mAb) or without (Iso, isotype control mAb) TL1A blockade. Numbers inside outlined areas indicate the percent of cells in the relevant gate (C, E, and G) and data are representative of two (G) or three (C and E) independent experiments. Each symbol represents an individual mouse and data are pooled from three (D and F) or two (B and H) independent experiments. Data are expressed as mean (±SEM) with P values determined by one-way ANOVA followed by Tukey’s multiple-comparisons test (B) or unpaired two-tailed Student’s t tests (D, F, and H): ns, not significant, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: Flow Cytometry, Expressing, Control, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: ILC9 cells have an increased capacity to initiate IL-5-dependent allergic airway inflammation. (A) Treatment schedule of naïve wild type (WT, C57BL/6J) mice by a single i.v. adoptive cell transfer of classical IL-33-activated ILC2s (ILC2) or IL-33/TL1A-activated ILC2s (ILC9). (B–H) Flow cytometry (B and D) and frequency of eosinophils (Gr1 low Siglec-F + CD11c − cells) among live CD45 + cells from BALF (C and F) or lung (E and G), and number of Red5 + ILC2s or ILC9s in total lung of mice (H), at day 7 after a single i.v. adoptive transfer of 5 × 10 5 ILC2s or ILC9s in separate host mice. Adoptively transferred ILC2s and ILC9s were prepared from Rag2 −/− mice ( Il5 +/+ cells) (B–E) or Red5 mice ( Il5 −/− cells) (F–H). Control mice received an intravenous injection of PBS. Red5 + cells indicate the activity of the Il5 promoter. Each symbol represents an individual mouse and data are representative (B and D) or pooled (C and E–H) from two independent experiments. (I–K) Live imaging of ILC2s and ILC9 cells in the lung. Lung intravital microscopy was performed 1–4 h after adoptive transfer of 6 × 10 5 of each cell type in the same host (green, classical IL-33-activated ILC2s-CFSE + ; red, IL-33/TL1A-activated ILC9 cells-CTO + ) (I). Imaging of the migratory behavior of ILC2s and ILC9 cells in the lung (J) and cell quantification from lung intravital microscopy data (K). Time-lapse images, 2 h after adoptive cell transfer (J). A maximum intensity projection of stitched images (2 × 2 tiles and 18 z-stack) is shown (K). Time in h/min/s. Scale bars: J, 20 μm; K, 100 μm. Lung intravital microscopy data are representative (J and K) or analyzed (K) from three adoptive transfer experiments on four mice. Data are expressed as mean (±SEM) with P values determined by paired two-tailed Student’s t test (K) or one-way ANOVA followed by Tukey’s multiple-comparisons test (C and E–H): ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: Flow Cytometry, Adoptive Transfer Assay, Control, Injection, Activity Assay, Imaging, Intravital Microscopy, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: TL1A is an epithelial alarmin that cooperates with IL-33 for initiation of allergic airway inflammation

doi: 10.1084/jem.20231236

Figure Lengend Snippet: Related to . Adoptively transferred ILC2s and ILC9s are equally recruited to the lung and exhibit an ameboid-like mode of migration. IL-33-activated ILC2s (CFSE/green), IL33/TL1A-activated ILC9s (CTO/red), blood vessels (Evans Blue/dark blue), and collagen fibers (second harmonic generation/light blue) were observed by lung intravital multiphoton imaging 2 h after intravenous adoptive transfer (6 × 10 5 cells). Time in h/min/s. Playback speed: 600.

Article Snippet: Cells were then directly blocked with 1% bovine serum albumin in PBS and incubated for 1 h at room temperature with mAbs to mouse TL1A (rat IgG1 mAb, clone 293327, 2 μg/ml, # MAB7441; RRID: AB_2206977; R&D Systems) or DDK (Flag) epitope (rabbit mAb, clone TA592569S, 1 μg/ml, # TA592569; Origene).

Techniques: Migration, Imaging, Adoptive Transfer Assay

Journal: Slas Discovery

Article Title: Identification of potent small molecule inhibitors of SARS-CoV-2 entry

doi: 10.1016/j.slasd.2021.10.012

Figure Lengend Snippet: High-throughput of ReFRAME, Pathogen Box, TargetMol and Cathepsin L drug libraries for SARS-CoV-2 antiviral compounds. A. Schematic of the spike protein of SARS-CoV-2. RBD: receptor binding domain. B. Schematic of the High-throughput assay. Compounds were pre-spotted in 1536-well plates. Next, 2000 HEK293T-ACE2 cells were added to each well and pre-incubated with each compound for 1 h, followed by infection with MLV reporter luciferase virus pseudotyped with the SARS-CoV-2 Spike protein (SARS2-S) or VSV-G protein (VSV-G). Luciferase was measured 48 h later. C. Summary of the ReFRAME library results. Conc.: concentration. D. Distribution of Z-Score for primary screens of each library. Scatter plot of Z_Score for all samples tested from the ReFrame library ( N = 1; circle) and other libraries ( N = 3; Cathepsin L: square; Pathogen Box: cross; TargetMol: filled circle). Total of 16,320 samples. Positive controls: orange; Negative control: cyan; Hit compounds: red; non-hit compounds: black. E. Summary of the 3 other libraries results. F. ReFrame library screening against different targets: SARS2-S, 3CLpro and PLpro. Venn diagram analysis of comparison between hits from SARS2-S entry, 3CLpro and PLpro assay against ReFRAME library results. There are 419 compounds that are SARS2-entry specific potential inhibitors. G. Robustness in terms of Z’ score of each screen for each library.

Article Snippet: Vero E6 cells were stained with Goat anti-Human Phycoerythrin-conjugated ACE2 Polyclonal Antibody (R&D Systems) for 30 min at 4 °C in dark.

Techniques: High Throughput Screening Assay, Binding Assay, Incubation, Infection, Luciferase, Virus, Concentration Assay, Negative Control, Library Screening, Comparison

Journal: Slas Discovery

Article Title: Identification of potent small molecule inhibitors of SARS-CoV-2 entry

doi: 10.1016/j.slasd.2021.10.012

Figure Lengend Snippet: Summary of the selected Cathepsin L, Pathogen box and TargetMol compounds in this study. Activity of the selected compounds against the different MLV pseudotyped viruses in HEK293-ACE2 cells and their respective cytotoxicity. Values for SARS2-S, VSV-G and toxicity are mean ± SEM of 2–4 independent experiments. TI: therapeutic index. * n = 1.

Article Snippet: Vero E6 cells were stained with Goat anti-Human Phycoerythrin-conjugated ACE2 Polyclonal Antibody (R&D Systems) for 30 min at 4 °C in dark.

Techniques: Activity Assay

Journal: Slas Discovery

Article Title: Identification of potent small molecule inhibitors of SARS-CoV-2 entry

doi: 10.1016/j.slasd.2021.10.012

Figure Lengend Snippet: Targets of the selected compounds and SARS-CoV-2 wild type infection. A. Description of the targets of the different hits from all the studied libraries. B. Antiviral activity of the 2 best hits in the SARS-CoV-2-induced CPE assay. Vero E6 cells treated with test compounds for two hours were infected with SARS-CoV-2 at an MOI of 0.05, then incubated for three days in the presence of compound. Cell viability (protection from virus-induced CPE) was measured with CellTiter-Glo. C and D. Antiviral effect was measured with a subset of Vero E6 cells expressing a low (C) and high (D) level of ACE2. E. Cytotoxicity of selected compounds in Vero E6 cells. Cytotoxicity was tested in the same conditions with cell culture media instead of the virus. F. Virus yield reduction activity of selected compounds. Vero E6 cells infected with SARS-CoV-2 at an MOI of 0.05 were cultured in the presence of test compound (5 µM) and the supernatant was harvested after 24 and 48 h of incubation. The Progeny virus was enumerated with a plaque assay using an Avicel overlay in fresh Vero E6 cells. N = 3 experiments were performed for infectivity assays and n = 2 for the cytotoxicity assays. **** P < 0.0001, Two-way ANOVA with Dunnett's multiple comparisons test against DMSO control.

Article Snippet: Vero E6 cells were stained with Goat anti-Human Phycoerythrin-conjugated ACE2 Polyclonal Antibody (R&D Systems) for 30 min at 4 °C in dark.

Techniques: Infection, Activity Assay, Incubation, Virus, Expressing, Cell Culture, Plaque Assay, Control

Journal: Slas Discovery

Article Title: Identification of potent small molecule inhibitors of SARS-CoV-2 entry

doi: 10.1016/j.slasd.2021.10.012

Figure Lengend Snippet: SR-914 “calpeptin” specifically blocks SARS-CoV entry. A. Its activity against SARS2-S in HEK293T-ACE2-TMPRSS2 cells. Cells were incubated with different concentrations of drugs, then infected with SARS2-S or VSV-G. Luciferase was measured 48 h later, using Bright-Glo. Shown is the mean ± SEM of n = 2 to 4 independent experiments. B. Time of drug addition experiment schematic. Infection was performed for 1 h with or without drugs, Vero CCL81 cells were then washed, and fresh media was added with or without drugs. C. Time of drug addition experiment result. SR-914 was used at 10 µM. E64d at 20 µM. Calp.: calpeptin = SR-914. NI: not infected. Shown is the mean ± SEM of 4 to 6 independent experiments. D. Luciferase complementation assay schematic. The reporter consists of a split Firefly luciferase protein connected by a cleavable peptide for the tested protease. Upon cleavage of the peptide, the luciferase protein undergoes dimerization for an active state. DnaE intein helps in this dimerization. E. Its activity against SARS2-S Entry, 3CLpro and PLpro. C-: negative control. C+; positive control. Shown is the mean ± SD of 3 independent experiments. One-way ANOVA followed by Tukey's post-test were used for statistical comparisons. *, P < 0.01; **, P < 0.001; ***, P < 0.0001.

Article Snippet: Vero E6 cells were stained with Goat anti-Human Phycoerythrin-conjugated ACE2 Polyclonal Antibody (R&D Systems) for 30 min at 4 °C in dark.

Techniques: Activity Assay, Incubation, Infection, Luciferase, Negative Control, Positive Control

Journal: Slas Discovery

Article Title: Identification of potent small molecule inhibitors of SARS-CoV-2 entry

doi: 10.1016/j.slasd.2021.10.012

Figure Lengend Snippet: Breath of activity of calpeptin against various SARS-CoVs. A. Its activity against SARS1-S in HEK293T-ACE2 cells. HEK293T-ACE2 cells were incubated with different concentrations of calpeptin, then infected with SARS1-S. Luciferase was measured 48 h later, using Bright-Glo. Shown is the mean ± SEM of n = 2 independent experiments. B. Schematic of the substituted residues in the S protein of the highest threat of SARS-CoV-2 strains. C. Evolution of the S protein residues at the position 417, 484, 501 and 614 from 2019 to February 2021. Modified figure from https://nextstrain. org/ncov/global?branchLabel=none& c =gt-S_417,484,501,614& l =clock. D. Activity of the new emergent variants. HEK293T-ACE2 cells were infected with different mutants of SARS2-S. The day after, a medium change was performed. Luciferase was measured 48 h later, using Bright-Glo. Shown is the mean ± SEM of n = 3 independent experiments. WT: wild type, SA: South Africa, UK: United Kingdom. E. Activity of calpeptin activity against crucial mutations present in the S protein of the new emergent strains. Similar experiment than D but calpeptin was added during infection and after medium change. Shown is the mean ± SEM of n = 2–5 independent experiments. Two-way ANOVA followed by Dunnett's post-test were used for statistical comparisons. *, P < 0.01; **, P < 0.001; ***, P < 0.0001.

Article Snippet: Vero E6 cells were stained with Goat anti-Human Phycoerythrin-conjugated ACE2 Polyclonal Antibody (R&D Systems) for 30 min at 4 °C in dark.

Techniques: Activity Assay, Incubation, Infection, Luciferase, Modification

Journal: Translational Oncology

Article Title: BEZ235 (PIK3/mTOR inhibitor) Overcomes Pazopanib Resistance in Patient-Derived Refractory Soft Tissue Sarcoma Cells

doi: 10.1016/j.tranon.2016.03.008

Figure Lengend Snippet: (A) Using high-throughput 3-dimensional culture system, the anti-tumor activity of 13-drug panel ( left upper panel ) was tested using PDC line. The results from 3-dimensional drug screening results were further confirmed with in vitro cell viability assay ( left lower panel ). The growth of tumor cells was significantly reduced by exposure to 1.0 μM AZD2014 compared with control (control versus AZD2014, mean growth = 100.0% vs 16.04%, difference = 83.96%, 95% CI = 70.01% to 97.92%, P = .0435). 1.0 μM BEZ235 profoundly inhibited tumor cell growth in vitro when compared to control (control versus BEZ235, mean growth = 100.0% vs 7.308%, difference = 92.69%, 95% CI = 78.87% to 106.5%, P < .0001). (B) The effects of AZD2014, BEZ235, lapatinib, LEE011, pazopanib on PI3K/AKT signaling in sarcoma PDC line were determined by immunoblotting analysis. Cells were treated with 1 μM of the indicated drugs for 72 h.

Article Snippet: Thirty micrograms of proteins were subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis on 10% gels and then electrotransferred to nitrocellulose membranes incubated with the following specific antibodies: Cyclin D1 (sc-718) (M-20) from Santa Cruz (USA) and PI3K (#4255) (p110α), phospho-mTOR (#2971) (Ser2448), mTOR (#2983) (7C10), phospho-Akt (#4060) (Ser473), Akt (#9272), phospho-ERK1/2 (#4370) (Thr202/Tyr204), ERK1/2 (#9102) from Cell Signaling Technologies (Beverly, MA, USA).

Techniques: High Throughput Screening Assay, Activity Assay, Drug discovery, In Vitro, Viability Assay, Control, Western Blot

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A Schematic representation of luciferase screening approach. Upper panel shows the four target predictions software used for in silico analysis. Bottom panel indicates the main steps performed in the high‐throughput screening. B Upper panel, sequence interaction of miR‐182‐3p with the target site of the wild type 3′UTR of TRF2 in human. Bottom panel, generation of mutant 3′UTR of TRF2 luciferase construct containing the deletion of target site for miR‐182‐3p. C–E Luciferase reporter assay in HeLa cells using the synthetic miR‐Control or miR‐182‐3p in combination with the wild type (C) or the mutant 3′UTR of TRF2 construct (D) or the wild type 3′UTR of TRF1 (E). F, G Western blotting for TRF2 expression in telomerase‐positive (HeLa, HCT116, MDA‐MB‐231, MDA‐MB‐436) and ALT‐positive (U2‐OS, Saos‐2) cells transiently transfected with miR‐Control or miR‐182‐3p. Upper panel shows the quantification of TRF2 expression. Bottom panel, representative images are shown, actin was used as loading control. H U2‐OS cells transiently transfected with the miR‐Control, miR‐182‐3p or miR‐182‐3p inhibitor were assayed by quantitative immunofluorescence for TRF2 3 days post‐transfection. Left panel, representative images. Scale bar: 10 μm. Right panel, quantification of TRF2 fluorescence intensity. a.f.u. arbitrary fluorescence units. N = number of analyzed nuclei. Red bar indicates mean value. I U2‐OS cells transfected as described in (H) were assayed by immunofluorescence combined with telomeric FISH. Left panel, representative images of co‐localizations between TRF2 and telomeres (white arrowheads). Scale bar: 10 μm. Right panel, co‐localizations were analyzed using ImageJ software. N = number of analyzed nuclei. Data information: For (C–G and I), data are shown as mean ± SD. Three independent experiments were performed ( n = 3). P values are determined by Student's t ‐test; for (H), P values are determined by Mann–Whitney t ‐test. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Luciferase, Software, In Silico, High Throughput Screening Assay, Sequencing, Mutagenesis, Construct, Reporter Assay, Western Blot, Expressing, Transfection, Immunofluorescence, Fluorescence, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A Results of high‐throughput luciferase screening performed in Hela cells using the wild type 3′UTR‐TRF2 vector in combination with each of the 54 miRNAs selected by in silico analysis. Three days post‐transfection, luciferase ratio (Renilla:Firefly) of each miRNA was calculated, the control miRNA was set “1.” Renilla:Firefly ratios < 1 indicate target specificity of candidate miRNAs for the 3′UTR of TRF2. miRNAs near to the ratio of 0.5 were considered for further analysis. Two biological replicates were performed. B HeLa cells transiently transfected with the indicated miRNAs (miR‐Control, miR‐182‐3p, miR‐519e‐5p, miR‐296‐3p) were assayed by western blotting. Upper panel, quantification of TRF2 expression. Bottom panel, representative images of TRF2, TRF1 and RAP1 are shown, actin was used as loading control. C Analysis of TRF2 mRNA expression performed by qPCR in four different cancer cell lines (HeLa, MDA‐MB‐231, MDA‐MB‐436, U2‐OS) 3 days post‐transfection with miR‐Control or miR‐182‐3p. The control miRNA was set “1.” Three independent experiments were performed. D, E Telomeric ChIP assay in MDA‐MB‐231 (D) and U2‐OS cells (E). Quantification of TRF2 enrichment at telomeric repeats, in the different conditions, is shown in the table under the respective figure. Alu probe and Rabbit IgG were used as negative control for the assay. Data information: For (A), data are presented as mean values. For (B, C), data are presented as mean values ± SD and Student t‐ test was used to calculate statistical significance. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: High Throughput Screening Assay, Luciferase, Plasmid Preparation, In Silico, Transfection, Western Blot, Expressing, Negative Control

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A MDA‐MB‐231 cells were transiently transfected with the indicated miRNAs or siRNA. The indicated DNA damage markers were assayed by western blotting. Actin was used as loading control. B Telomeric DNA FISH performed in MDA‐MB‐231 transiently transfected with the indicated miRNAs. Telomere length was measured by TLF software and indicated as arbitrary fluorescence unit (a.f.u). N = number of analyzed nuclei. Black bar indicates mean value. C DNA damage markers were assayed by western blotting in HeLa cells. Actin was used as loading control. D Immunofluorescence analysis of γH2AX combined with a telomeric FISH probe (TIFs) was performed in HeLa cells transfected with the indicated miRNAs or siRNAs. Co‐localizations of γH2AX with telomeres are indicated as mean number of TIFs per nucleus. E Representative images and enlargements of co‐localizations of experiment described in D. F Immunofluorescence analysis of γH2AX combined with a SatIII FISH probe (PIFs) was performed in HeLa cells transfected with the indicated miRNAs or siRNAs. The γH2AX‐positive cells with ≥ 1 PIFs per nucleus were analyzed. G Representative images of co‐localizations relative to the experiment described in (F). H, I MDA‐MB‐231 and HeLa cells over‐expressing TRF2 or an empty vector (pBabe) were transiently transfected with miR‐Control or miR‐182‐3p. TRF2, pATM and γH2AX expression were assayed by western blotting. Actin was used as loading control. Data information: For (D) and (F), data are presented as mean values ± SD. Three independent replicates were performed. Scale bar: 10 μm. At least 60 nuclei were analyzed in (D) and (F). A Student t‐ test was used to calculate statistical significance. For (B), P values are determined by Mann–Whitney t ‐test. All the experiments were performed 3 days post‐transfection with the indicated miRNAs or siRNAs. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Transfection, Western Blot, Software, Fluorescence, Immunofluorescence, Expressing, Plasmid Preparation, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: Immunofluorescence analysis of γH2AX combined with telomeric FISH (TIFs) was performed in MDA‐MB‐231 cells transfected with the indicated miRNAs or siRNAs. The mean number of TIFs per nucleus was analyzed. Representative images and enlargements of co‐localizations (white arrowheads) relative to the experiment described in (A). Scale bar: 10 μm. Immunofluorescence analysis of γH2AX combined with a SatIII FISH probe (PIFs) was performed in MDA‐MB‐231 cells transfected with the indicated miRNAs or siRNAs. The γH2AX‐positive cells with ≥ 1 PIFs per nucleus were analyzed. Representative images of co‐localizations (white arrowheads) relative to the experiment described in (C). Scale bar: 10 μm. Quantification of TIFs in MDA‐MB‐231 cells over‐expressing TRF2 or an empty vector (pBabe), transfected with indicated miRNAs. The mean number of TIFs per nucleus was quantified. Representative images and enlargements relative to the experiment described in (E). White arrowheads indicate co‐localizations events. Scale bar: 10 μm. Quantification of PIFs in MDA‐MB‐231 cells over‐expressing TRF2 or an empty vector (pBabe), transfected with indicated miRNAs. The γH2AX‐positive cells with ≥ 1 PIFs per nucleus were analyzed. Representative images relative to the experiment described in (G). White arrowheads indicate co‐localizations events. Scale bar: 10 μm. Data information: For (A, C, E, G) data are shown as mean ± SD. Three independent experiments were performed ( n = 3). P values are determined by unpaired two‐tailed t‐ test. At least 60 nuclei were analyzed for each experimental condition. All the experiments were performed 3 days post‐transfection with the indicated miRNAs or siRNAs. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Immunofluorescence, Transfection, Expressing, Plasmid Preparation, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A, B MDA‐MB‐436 and MDA‐MB‐231 cells underwent two rounds of transfection with miR‐Control, miR‐182‐3p or miR‐182‐3p inhibitor. Starting from the day of the second transfection, cell confluence was monitored by Incucyte every 24 h up to a maximum of 3 days. The percentage of cell confluence was analyzed. C, D Cell number of MDA‐MB‐436 (C) and MDA‐MB‐231 (D) cells and TRF2 expression were analyzed by automatic cell count and by western blotting at the end of the experiment described in (A) and (B). Actin was used as loading control. E Two‐dimensional scatter plots of Annexin V analysis performed in MDA‐MB‐436 at the end of the second cycle of transfection with miR‐Control, miR‐182‐3p or miR‐182‐3p inhibitor. Red boxes indicate early and late apoptotic cells. F Quantification of Annexin V‐positive cells (%) of experiment described in (E). G Two‐dimensional scatter plots of Annexin V analysis performed in MDA‐MB‐231 as described in (E). H Quantification of Annexin V‐positive cells (%) of experiment described in (G). I, J MDA‐MB‐436 cells over‐expressing TRF2 or an empty vector (pBabe) were transiently transfected with indicated miRNAs and cell count (I) or apoptosis (J) analysis was performed 72 h post‐transfection. Data information: For (A, B) data are shown as mean ± SEM. For (C, D, F, H, I, J), data are shown as mean ± SD. For (A–D) and (I), three independent experiments were performed ( n = 3). P values are determined by unpaired two‐tailed t‐ test. For (F), (H) and (J), two different biological replicates were performed. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Transfection, Expressing, Cell Counting, Western Blot, Plasmid Preparation, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A Western blotting for TRF2 expression in BJ cells transiently transfected with miR‐Control or miR‐182‐3p. The graph represents the quantification of three independent experiments. Representative images are shown, Actin was used as loading control. Unspecific bands are indicated with (*). B, C Mean of γH2AX foci per nucleus was analyzed in BJ cells 72 h post‐transfection with the indicated miRNAs. Representative images of γH2AX foci are shown in (C). D Immunofluorescence analysis of γH2AX combined with a telomeric FISH probe (TIFs) was performed in BJ cells 72 h post‐transfection with the indicated miRNAs. Left panel: The mean number of TIFs per nucleus was analyzed. Right panel: Representative images and enlargements of co‐localizations. E Cell number of BJ cells was analyzed by automatic cell count at the end of the second round of transfection with miR‐Control or miR‐182‐3p. F FACS analysis to evaluate cell cycle progression by Propidium Iodide (PI) staining in BJ cells treated as indicated in (E). G β‐Galactosidase assay in BJ cells after two rounds of transfection with mimic miR‐Control or miR‐182‐3p. Left panel: Analysis of β‐galactosidase‐positive cells. Right panel: Representative images. H–J IL‐6 (H), CXCL1 (I), IL‐8 (J) factors were analyzed by ELISA to evaluate the senescence‐associated secretory phenotype (SASP) in BJ cells treated as indicated in (G). Data information: For (A, B, D, E and G–J), a student t‐ test was used to calculate statistical significance. Scale bars (10 μm). P values are indicated. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Western Blot, Expressing, Transfection, Immunofluorescence, Cell Counting, Staining, Enzyme-linked Immunosorbent Assay

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A TRF2 and γH2AX expression after two rounds of transfection with the indicated miRNAs, was analyzed by western blotting in MCF10A cells. Actin was used as loading control. B–E The mean number of γH2AX foci (B) and TIFs (D) per nucleus were analyzed 72 h post‐transfection with the indicated mimic miRNAs in MCF10A cells. Representative images (C) and (E) are referred to the experiment showed in (B) and (D) respectively. F, G Cell confluence (F) of MCF10A was monitored by Incucyte, every 24 h starting from the day of the second transfection, and cell number (G) was counted at the end of experiment (day 4). H–I Cell cycle progression analysis by PI staining (H) and cell death analysis by Annexin V assay (I) were performed in MCF10A upon two rounds of transfection with the indicated miRNAs. J β‐Galactosidase assay in MCF10A cells after two rounds of transfection with mimic miR‐Control or miR‐182‐3p. Left panel: Analysis of β‐galactosidase‐positive cells. Right panel: Representative images. Data information: Panels (B, D, F, G, J) data are presented as mean values ± SD. A Student t‐ test was used to calculate statistical significance. P values are indicated. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Expressing, Transfection, Western Blot, Staining, Annexin V Assay

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A, B MDA‐MB‐231 (A) and MDA‐MB‐436 (B) tumor xenografts were treated with LNPs‐empty, LNPs‐miR‐Control or by LNPs‐miR‐182‐3p when the tumors became palpable. Mice were treated 6 times by intravenous tail vein injections with 20 μg of LNPs‐miR‐Control, LNPs‐miR‐182‐3p or equivalent volume of LNPs‐empty as indicated in the scheduling. The mean of tumor volumes ( n = 5 per group) is shown. C, D Tumors from mice treated in (A) and (B) were processed to measure miR‐182‐3p expression by TaqMan qPCR. E Representative images of IHC analysis of the indicated markers on tumor samples from mice bearing MDA‐MB‐231 human breast cancer xenografts. Scale bar: 50 μm. F The histograms show the expression of TRF2, calculated as immunoreactivity score (IRS) by IHC, and the count of positive cells to γH2AX, TUNEL or CD31 staining. The analyses were performed on three mice per group, and the points represent the number of field analyzed for each condition. G, H Luminescent MDA‐MB‐436 cells were injected into the brain and monitored by IVIS imaging system. After 1 week from implant, treatment with LNPs‐miR‐Control and LNPs‐miR‐182‐3p was performed as indicated in (A) and (B). Representative images from in vivo (upper panel) or ex‐vivo (bottom panel) brain tumors are shown in (G). Boxplots (H) show the measurement of photons for each brain tumor ( n = 5 per group) acquired at the indicated times. Data information: For (A, B, F), data are shown as mean ± SD. For (C, D, H), the line in the middle of the box plot denotes a median value, the limits of box represent the interquartile range (25 th to 75 th percentiles), while, the whiskers denote the minimum to maximum values. For (A–D) and (H), P values are determined by unpaired two‐tailed t‐ test; for (F), P values are determined by Mann–Whitney t ‐test. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Expressing, TUNEL Assay, Staining, Injection, Imaging, In Vivo, Ex Vivo, Two Tailed Test, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: The organs (brain, liver, kidney) taken from mice, previously engrafted with MDA‐MB‐231 cells and treated with LNPs‐empty, LNPs‐miR‐Control or LNPs‐miR‐182‐3p, were assayed for miR‐182‐3p expression by TaqMan qPCR. Representative images show IHC analysis on tumor samples, from mice bearing MDA‐MB‐436 human breast cancer xenografts, with the indicated markers. Scale bar: 50 μm. The histograms show the expression of TRF2 indicated as immunoreactivity score (IRS) and the percentage of positive cells to γH2AX, TIUNEL or CD31 staining in MDA‐MB‐436 xenografts. Three mice per group were analyzed, the points represent the number of field analyzed for each condition. Data information: For (A, C), data are presented as mean values ± SD. Statistical significance using unpaired (A) or Mann–Whitney t‐ test (C) was calculated. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Expressing, Staining, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A, B PDTCs #1 and #2 underwent two rounds of transfection with miR‐Control or miR‐182‐3p. Three days after the second transfection, miR‐182‐3p and TRF2 expression were analyzed by TaqMan qPCR and western blotting, respectively. Actin was used as loading control. C, D Left panel, area of each PDTCs was measured by ImageJ. Right panel, representative images are shown. Scale bar: 50 μm. At least 85 3D cells were analyzed for each experimental condition. E NSG mice implanted with breast PDTX (#2) were treated with LNPs‐empty, LNPs‐miR‐Control or LNPs‐miR‐182‐3p as indicated in the scheduling. Caliper measurement of tumors was taken at the indicated days. The mean of tumor volumes ( n = 5 per group) is shown. F miR‐182‐3p expression of tumors from mice treated in (E) was assayed by TaqMan qPCR. G Representative images of IHC analysis of the indicated markers from tumors of the experiment showed in (E). Scale bar: 50 μm. H The histograms show the expression levels of TRF2 measured as immunoreactivity score (IRS), the percentage of positive cells to γH2AX and TUNEL. The analysis was performed on three mice per group, the points represent the number of field analyzed for each condition. Data information: For (A–F) and (H), data are shown as mean ± SD. For (A–F), P values are determined by unpaired two‐tailed t‐ test; for (H), P values are determined by Mann–Whitney t ‐test. For the experiments showed in (A, B) and (C, D) two or three biological replicates were performed, respectively. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Transfection, Expressing, Western Blot, TUNEL Assay, Two Tailed Test, MANN-WHITNEY

Journal: EMBO Molecular Medicine

Article Title: MiR ‐182‐3p targets TRF2 and impairs tumor growth of triple‐negative breast cancer

doi: 10.15252/emmm.202216033

Figure Lengend Snippet: A Representative images of intestine sections from mice previously treated with LNPs‐Empty or LNPs‐miR‐182‐3p. H&E staining (scale bar: 200 μm) and IHC analysis with TRF2 or γH2AX antibodies are shown (scale bar: 50 μm). B, C Quantification of TRF2 expression as immunoreactivity score (IRS) (B) and of γH2AX‐positive cells (%) (C) on intestine samples. D Representative H&E (scale bar: 200 μm), TRF2 and γH2AX images of skin samples corresponding to LNPs‐Empty or LNPs‐miR‐182‐3p treated animals (scale bar: 50 μm). E, F Quantification of TRF2 expression as immunoreactivity score (IRS) (E) and of γH2AX‐positive cells (%) (F) on skin samples. G Representative H&E (scale bar: 200 μm), TRF2 and γH2AX images of bone marrow samples corresponding to LNPs‐Empty or LNPs‐miR‐182‐3p treated animals (scale bar: 50 μm). H, I Quantification of TRF2 expression as immunoreactivity score (IRS) (H) and of γH2AX‐positive cells (%) (I) on bone marrow samples. Data information: For (B, C, E, F, H, I), data are shown as mean ± SD. A Mann–Whitney test t‐ test was used to calculate statistical significance. Four mice per group were analyzed, the points represent the number of field analyzed for each condition. Source data are available online for this figure.

Article Snippet: The antibody used for the immunoprecipitation is the rabbit anti‐TRF2 (NB110‐57130, Novus) and IgG Rabbit (Bethyl) were used as negative control.

Techniques: Staining, Expressing, MANN-WHITNEY

Journal: Cancer Discovery

Article Title: NSD3–NUT Fusion Oncoprotein in NUT Midline Carcinoma: Implications for a Novel Oncogenic Mechanism

doi: 10.1158/2159-8290.cd-14-0014

Figure Lengend Snippet: Figure 1. A novel NSD3–NUT fusion is identifi ed in NMC. A, histology of the NMC from which the 1221 cell line was derived reveals a very poorly differentiated tumor (magnifi cation, ×400). B, IHC of the tumor using the anti-NUT monoclonal antibody C52 (magnifi cation, ×400). C, RNA-sequencing reads spanning the junction of NSD3 and NUT . D, immunoblot of three NMC cell lines and 293T control cells stained with AX.1 polyclonal antibody to NUT. E, immunoblot of the 1221 cell line 48 hours following transfection with control (CTRL), NSD3 , and NUT siRNAs stained with the AX.1 antibody to NUT. F, NSD3–NUT dual-color bring-together FISH assay (magnifi cation, ×1,000) using bacterial artifi cial chromosome (BAC) probes telomeric (3′) to NUT (green), and BAC probes centromeric (5′) to NSD3 (red) as depicted in the chromosomes 8 and 15 ideograms. Yellow arrows, NSD3–NUT fusions. G, gel electrophoresis of PCR of TC-797 and 1221 cell lines with (+) and without (−) reverse transcriptase reaction. H, schematic of the NSD3–NUT predicted encoded protein in comparison with NSD3, NUT, and BRD4–NUT. PWWP, Pro–Trp–Trp–Pro motif; PHD, plant homeo domain; SET, Drosophila Su(var)3-9 and ‘Enhancer of zeste’; C/H, Cys-His; NES, nuclear export signal sequence; Bromo, bromodomain. Arrows, breakpoints. I, NSD3 dual-color split-apart FISH assay using BAC probes fl anking NSD3 , as depicted in the chromosome 8 ideogram, depicted in three NMCs, not including 1221, desig- nated cases 1–3. All photomicrographs are of identical magnifi cation (×1,000).

Article Snippet: Immunohistochemical stains were performed using anti-NUT antibody (1:100, rabbit monoclonal clone C52; Cell Signaling Technology), anti-involucrin antibody (1:12,000; Sigma-Aldrich), and Ki-67 (MIB-1 clone; Dako; ref. 2 ).

Techniques: Derivative Assay, RNA Sequencing, Western Blot, Control, Staining, Transfection, Nucleic Acid Electrophoresis, Reverse Transcription, Comparison, Sequencing

Journal: Cancer Discovery

Article Title: NSD3–NUT Fusion Oncoprotein in NUT Midline Carcinoma: Implications for a Novel Oncogenic Mechanism

doi: 10.1158/2159-8290.cd-14-0014

Figure Lengend Snippet: Figure 3. Wild-type NSD3 is required for the blockade of differentiation in BRD4–NUT-expressing NMC cells. A, immunoblots of BRD4–NUT-positive NMC cell lines TC-797, PER-403, and 8645 120 hours following transfection with control and NSD3 siRNAs stained with the terminal squamous differen- tiation marker involucrin, using GAPDH as loading control. B, representative photomicrographs of TC-797s 120 hours following transfection with either control or NSD3 siRNAs stained either with H&E for morphology, or involucrin IHC. All photographs are of identical magnifi cation (×400). C, qRT-PCR of NSD3 levels 24 hours following transfection of control or NSD3 siRNAs. Primers were either 5′ of the breakpoint (NSD3–5′ primers), or 3′ of the break- point (NSD3-3′ primers) with NUT. Results are of a single biologic replicate performed in triplicate. Error bars, the mean ± SD of the triplicate wells. D, proliferation assay (Ki-67 fraction) comparing BRD4–NUT-positive TC-797, 8645, and PER-403 NMC cells transfected with control and NSD3–6 siRNAs. Three hundred cells were counted per cell block. E, 797TRex cells induced to express FLAG-tagged NLS-ET domain of BRD4 for 120 hours. Immu- noblot was performed with anti-involucrin (Inv), anti-FLAG, or anti-GAPDH (left). Cell block preparations were H&E stained, or subjected to involucrin IHC (right). All photographs are of identical magnifi cation (×400). F, cell viability assay (CellTiter-Glo) of 797TRex, 293TRex, and U2OSTRex cells induced to express FLAG-tagged NLS-ET domain for 120 hours. Results are the average of three biologic replicates, each performed in quadruplicate and normal- ized to the negative control (ethanol vehicle control) for each cell line. Error bars, the mean ± SD of the three biologic replicates. Immunoblot demonstrat- ing NLS-FLAG-ET expression was stained with anti-FLAG or anti-GAPDH (right).

Article Snippet: Immunohistochemical stains were performed using anti-NUT antibody (1:100, rabbit monoclonal clone C52; Cell Signaling Technology), anti-involucrin antibody (1:12,000; Sigma-Aldrich), and Ki-67 (MIB-1 clone; Dako; ref. 2 ).

Techniques: Expressing, Western Blot, Transfection, Control, Staining, Marker, Quantitative RT-PCR, Proliferation Assay, Blocking Assay, Viability Assay, Negative Control

Journal: Cancer Discovery

Article Title: NSD3–NUT Fusion Oncoprotein in NUT Midline Carcinoma: Implications for a Novel Oncogenic Mechanism

doi: 10.1158/2159-8290.cd-14-0014

Figure Lengend Snippet: Figure 4. The N-terminus of NSD3 associates with BRD4 and BRD4–NUT. A, immunofl uorescence microscopy of 797TRex cells induced to express the HA-tagged portion of NSD3 included in NSD3–NUT (NSD3Tr) for 24 hours stained with anti-NUT monoclonal antibody (red), and anti-HA monoclonal anti- body (green). B, immunoblot of anti-HA immunoprecipitations (IP) of tet-repressor–positive C33A cell (C33A-6TR) lysates following induction of expres- sion of HA-tagged NSD3 variants: HA-NSD3 (full-length), HA-NSD3–NUT, and HA-NSD3-tr (NSD3 portion of the NSD3–NUT fusion protein). Indicated proteins were detected using anti-HA and anti-Brd4 antibodies. The smaller bands are degraded protein. C, immunoblot of anti-HA immunoprecipitations of C33A-6TR lysates following induction of expression of HA-tagged NUT, BRD4, and BRD4–NUT constructs stained with anti-HA, anti-NSD3, anti-p300, and anti-actin antibodies. To identify the NSD3-specifi c bands, lysates from TC-797s subjected to siRNA knockdown of NSD3 are shown. D, immunoblot of 797TRex lystes 120 hours following induction of expression of BioTAP-tagged NLS fusion construct of NSD3Tr stained with anti-involucrin, anti-PAP (recognizes the protein A moiety of the BioTAP tag), and anti-GAPDH antibodies.

Article Snippet: Immunohistochemical stains were performed using anti-NUT antibody (1:100, rabbit monoclonal clone C52; Cell Signaling Technology), anti-involucrin antibody (1:12,000; Sigma-Aldrich), and Ki-67 (MIB-1 clone; Dako; ref. 2 ).

Techniques: Microscopy, Staining, Western Blot, Expressing, Construct, Knockdown

Journal: Cancer Discovery

Article Title: NSD3–NUT Fusion Oncoprotein in NUT Midline Carcinoma: Implications for a Novel Oncogenic Mechanism

doi: 10.1158/2159-8290.cd-14-0014

Figure Lengend Snippet: Figure 5. BRD4–NUT foci are dependent on NSD3. A, immunofl uores- cence microscopy of TC-797 cells 24 hours following transfection with control or NSD3–6 siRNAs stained with monoclonal antibody to NUT. All photographs are of identical magnifi cation (×1,000). B, quantitation of BRD4–NUT foci was performed in triplicate and the average of the three experiments is shown. Error bars, the mean ± SD of triplicate experi- ments. *, P < 0.005. C, immunoblot of TC-797 lysates 24 hours following transfection with control, NSD3–6 , or NUT siRNAs stained with anti-NUT polyclonal antibody, AX.1.

Article Snippet: Immunohistochemical stains were performed using anti-NUT antibody (1:100, rabbit monoclonal clone C52; Cell Signaling Technology), anti-involucrin antibody (1:12,000; Sigma-Aldrich), and Ki-67 (MIB-1 clone; Dako; ref. 2 ).

Techniques: Microscopy, Transfection, Control, Staining, Quantitation Assay, Western Blot

Journal: Cancer Discovery

Article Title: NSD3–NUT Fusion Oncoprotein in NUT Midline Carcinoma: Implications for a Novel Oncogenic Mechanism

doi: 10.1158/2159-8290.cd-14-0014

Figure Lengend Snippet: Figure 6. NSD3–NUT can replace the function of BRD4–NUT to block differentiation. A, H&E and anti-involucrin IHC micrographs of 797TRex cells with tetracycline (ON), or treated with vehicle (OFF) to express NSD3–NUT 120 hours following transfection with either control or NUT 3′-UTR siRNA. All photographs are of identical magnifi cation (×400). B, immunoblots using lysates corresponding to the experiment in A were performed for the differen- tiation marker, involucrin, NSD3–NUT, and BRD4–NUT using antibodies to NUT. C, quantifi cation of immunohistochemical Ki-67 proliferation fraction of 797TRex cells induced to express NSD3–NUT 120 hours following transfection with either control or NUT 3′-UTR siRNA as in A. Results are the average of three biologic replicates performed using the 384-well high-throughput assay as in Fig. 2A , each performed in triplicate. Error bars, the mean ± SD of the three biologic replicates. *, P < 0.0001.

Article Snippet: Immunohistochemical stains were performed using anti-NUT antibody (1:100, rabbit monoclonal clone C52; Cell Signaling Technology), anti-involucrin antibody (1:12,000; Sigma-Aldrich), and Ki-67 (MIB-1 clone; Dako; ref. 2 ).

Techniques: Blocking Assay, Transfection, Control, Western Blot, Marker, Immunohistochemical staining, High Throughput Screening Assay

Journal: Molecular Biology of the Cell

Article Title: Rab18 is not necessary for lipid droplet biogenesis or turnover in human mammary carcinoma cells

doi: 10.1091/mbc.E18-05-0282

Figure Lengend Snippet: Rab18 localizes distinctly to LDs and the ER in SUM159 cells. (A) Overexpressed GFP-Rab18 localizes to LDs (LipidTox) (white arrowheads) and the ER (mCherry-ER3), and localization depends on GTP state (white arrows). SUM159 cells coexpressing mCherry-ER3 and GFP-tagged WT Rab18, GDP-bound Rab18(S22N) mutant, or GTP-bound Rab18(Q67L) mutant were incubated with OA for 0 or 18 h and imaged with spinning disk confocal. Scale bar 5 µm and for inlay 1 µm. (B) Rab18 localizes to the ER and LD structures. SUM159 cells co-expressing mCherry-ER3 and GFP-Rab18 were incubated with oleic acid for 18 h and imaged by SIM. Max projections of 1.25-µm stacks are shown. Scale bars, 1 µm. (C) Quantification of Rab18 signal distribution in SIM images. n = 5 fields. (D) Rab18 was detected in LD fractions and total cell lysates of SUM159 cells. LD fractions and cell lysates isolated from SUM159 cells after 18 h oleic acid were analyzed by mass spectrometry to detect proteins on LDs compared with total lysate. ND = not detected.

Article Snippet: We used rabbit polyclonal antibodies against Rab18 (Proteintech; 11304-1-AP), ATGL (CST; 2138S), Calnexin (Enzo: ADI-SPA-860), reticulon 4 (Santa Cruz; Nogo N18 SC11027), and glyceraldehyde 3-phos phate dehydrogenase (GAPDH) (Santa Cruz Biotechnology; sc-25778).

Techniques: Mutagenesis, Incubation, Expressing, Isolation, Mass Spectrometry

Journal: Molecular Biology of the Cell

Article Title: Rab18 is not necessary for lipid droplet biogenesis or turnover in human mammary carcinoma cells

doi: 10.1091/mbc.E18-05-0282

Figure Lengend Snippet: Rab18 deletion does not affect ER morphology. (A) Sequence analysis of Rab18 KO clones A and B. CRISPR/Cas9-mediated genome editing of the Rab18 locus introduces early stop codons at exons 4 (clone A) and 5 (clone B). (B) qPCR data reveal decreased Rab18 mRNA levels by 98 and 96% in Rab18KO-A and –B, respectively, compared with WT control. WT vs. Rab18KO-A* in gray, WT vs. Rab18KO-B* in black. (C) No Rab18 protein is detected in knockout clones by Western blot. Expression levels of Rab18 protein in WT and Rab18 KO cells were analyzed by Western blot with an antibody against endogenous Rab18. No detectable Rab18 protein was found in the Rab18KO-A or Rab18KO-B. (D) Rab18 peptide fragments were not detected by mass spectrometry in Rab18KO-A. WT SUM159 cell lysates and Rab18KO-A cell lysates were analyzed by mass spectrometry with sequence coverage of 68.4% for Rab18. (E) ER morphology in Rab18 KO clones is similar to WT cells. Cells were transfected with GFP-ERox to analyze general ER morphology. Separately, cells were fixed and probed with Reticulon 4 (Rtn4) antibody to visualize ER tubules. Scale bar 5 µm and for inlay 1 µm.

Article Snippet: We used rabbit polyclonal antibodies against Rab18 (Proteintech; 11304-1-AP), ATGL (CST; 2138S), Calnexin (Enzo: ADI-SPA-860), reticulon 4 (Santa Cruz; Nogo N18 SC11027), and glyceraldehyde 3-phos phate dehydrogenase (GAPDH) (Santa Cruz Biotechnology; sc-25778).

Techniques: Sequencing, Clone Assay, CRISPR, Control, Knock-Out, Western Blot, Expressing, Mass Spectrometry, Transfection

Journal: Molecular Biology of the Cell

Article Title: Rab18 is not necessary for lipid droplet biogenesis or turnover in human mammary carcinoma cells

doi: 10.1091/mbc.E18-05-0282

Figure Lengend Snippet: LD biogenesis is not affected in Rab18 KO cells. (A) LD morphology is similar in Rab18KO clones and WT cells with oleic acid incubation. Representative images of WT and Rab18 KO cells prestarved for 5 h before addition of oleic acid and after 24 h of oleic acid incubation. Scale bar 5 µm and for inlay 1 µm. (B) Rab18 KO average BODIPY object area and number are slightly smaller than WT after 24 h oleic acid incubation. WT and Rab18 KO cells were incubated with oleic acid for indicated time points, fixed, and imaged by high-throughput microscopy. Average BODIPY object area and number per cell per field (>five cells per field to obtain representative measurements) were quantified per condition. n > 27 fields/point. WT vs. Rab18KO-A* in gray; WT vs. Rab18KO-B* in black. (C) Rab18KO and WT average object area vs. number 90% confidence intervals overlap at 2 and 24 h oleic acid incubation. Average object number plotted against average object area per cell per field with 2 and 24 h oleic acid. Ellipses represent 90% confidence intervals. (D, E) Rab18KO clones have similar synthesis of TG, PE, and PC as WT cells with oleic acid incubation. Incorporation of [ 14 C] oleate into triglycerides (TGs), phophatidylethanolamine (PE), and phosphatidycholine (PC) were measure over time. Lipids were extracted from cells and separated by TLC. Representative autoradiographs of three replicates per genotype are shown (D). (E) Quantified TG, PC, and PE levels are similar between Rab18KO clones and WT cells over time. TLC plates were developed and incorporation of [ 14 C] oleate into TGs, PE, and PC was quantified using Fiji. Data presented are normalized CPM to mg/ml protein and relative to WT at t = 0 h. n = 3.

Article Snippet: We used rabbit polyclonal antibodies against Rab18 (Proteintech; 11304-1-AP), ATGL (CST; 2138S), Calnexin (Enzo: ADI-SPA-860), reticulon 4 (Santa Cruz; Nogo N18 SC11027), and glyceraldehyde 3-phos phate dehydrogenase (GAPDH) (Santa Cruz Biotechnology; sc-25778).

Techniques: Clone Assay, Incubation, High Throughput Screening Assay, Microscopy

Journal: Molecular Biology of the Cell

Article Title: Rab18 is not necessary for lipid droplet biogenesis or turnover in human mammary carcinoma cells

doi: 10.1091/mbc.E18-05-0282

Figure Lengend Snippet: Rab18 deletion does not affect TG turnover. (A) LDs are degraded similarly in Rab18KO clones and WT cells with starvation. Representative images of WT, Rab18KO-A, and Rab18KO-B cells after 12 h OA loading ( t = 0) and 24 or 48 h of starvation. LDs stained with BODIPY 493/503 and nuclei with Hoechst. Scale bar 5 µm and inlay 1 µm. (B) Rab18KO average BODIPY object area and number are similar to WT after 48 h starvation. WT and Rab18KO cells were incubated with oleic acid for indicated time points as in A. Cells were fixed and imaged by high-throughput microscopy. Average BODIPY object area and number per cell per field (>5 cells per field) was quantified per condition. n > 7 fields. WT vs. Rab18KO-A * in gray; WT vs. Rab18KO-B * in black. (C) Rab18KO clones have similar synthesis of TG and PE and less PC than WT cells with starvation. WT and Rab18KO cells were incubated with [ 14 C] oleic acid for 18 h, followed by starvation for increasing time. Total lipids were extracted at each time point and separated by TLC to detect radiolabeled TG, PE, and PC levels. Representative autoradiographs of three replicates per genotype. (D) Quantified TG and PE levels are similar, and PC levels are less between Rab18KO clones and WT cells over time with starvation. TG, PC, and PE signals were quantified from TLC plates using Fiji. Data presented are normalized CPM to mg/ml protein and relative to WT at t = 0 h. n = 3 biological replicates. (E) Rab18KO clones have decreased free fatty acid release over time with starvation. The [ 14 C]-labeled free fatty acid release measured over time with starvation after WT; Rab18KO-A, and Rab18KO-B cells were incubated with [ 14 C] oleic acid for 18 h. n = 3 biological replicates.

Article Snippet: We used rabbit polyclonal antibodies against Rab18 (Proteintech; 11304-1-AP), ATGL (CST; 2138S), Calnexin (Enzo: ADI-SPA-860), reticulon 4 (Santa Cruz; Nogo N18 SC11027), and glyceraldehyde 3-phos phate dehydrogenase (GAPDH) (Santa Cruz Biotechnology; sc-25778).

Techniques: Clone Assay, Staining, Incubation, High Throughput Screening Assay, Microscopy, Labeling

Journal: Journal of Virology

Article Title: A New Family of Small-Molecule CD4-Mimetic Compounds Contacts Highly Conserved Aspartic Acid 368 of HIV-1 gp120 and Mediates Antibody-Dependent Cellular Cytotoxicity

doi: 10.1128/JVI.01325-19

Figure Lengend Snippet: High-throughput screening of small molecules for their ability to expose the coreceptor binding site. (A) A cell-based ELISA (CBE) was adapted to screen a library comprising ∼108,000 small molecules. In the assay, HOS cells expressing HIV-1JR-FL EnvΔCT were plated in a 384-well-plate format. Small molecules or, as a positive control, sCD4 were added to expose the HIV-1JR-FL EnvΔCT epitope that can be recognized by the CD4i antibody 17b. 17b binding was detected by a horseradish peroxidase (HRP)-conjugated second antibody, and HRP enzyme activity was measured by Western Lightning oxidizing and luminal reagents. (B) 17b binding in the presence of sCD4 was set as the control, small molecules that enhanced 17b binding above 25% of the one induced by sCD4 were retested in quadruplicate, and only one molecule (UM0059920) was deemed a true positive. (C) UM0059920 is a racemic mixture. (D) Addition of (S)-MCG-II-153 but not (R)-MCG-II-156 enhances 17b binding to levels similar to those of UM0059920 in the CBE. Data shown are mean relative light unit (RLU) values ± standard deviations (SD) from three independent experiments performed in quadruplicate, with the signal obtained from wells transfected with an empty pcDNA3.1 plasmid (no Env) subtracted, normalized to Env levels as determined by 2G12 binding. Statistical significance was evaluated by using an unpaired t test (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant).

Article Snippet: A horseradish peroxidase (HRP)-conjugated antibody specific for the Fc region of human IgG (Pierce) was then incubated with the samples for 45 min at room temperature.

Techniques: High Throughput Screening Assay, Binding Assay, In-Cell ELISA, Expressing, Positive Control, Activity Assay, Western Blot, Transfection, Plasmid Preparation

Journal: EMBO Molecular Medicine

Article Title: Integrative analysis of cell state changes in lung fibrosis with peripheral protein biomarkers

doi: 10.15252/emmm.202012871

Figure Lengend Snippet: The scatter plots show the positive correlation of CFHR1 in BALF (MS‐intensity, x ‐axis) with meta lung function ( y ‐axis). UMAP visualizes embedding of single‐cells colored by gene expression for CRTAC1 , which is specifically expressed in alveolar type‐2 (AT2), Club and lymphatic endothelial (Lymp_EC) cells. Relative expression level of CRTAC1 across human organs. The box plots illustrate differences in mRNA detection for CRTAC1 in alveolar epithelial cells from fibrosis patients compared to control samples across the three indicated patient cohorts (Chicago cohort: ILD n = 9, controls n = 8; Nashville cohort: ILD n = 20, controls n = 10; Munich cohort: ILD n = 3, controls n = 11). The boxes represent the interquartile range, the horizontal line in the box is the median, and the whiskers represent 1.5 times the interquartile range. Relative gene expression levels of CRTAC1 in GSE47460. Dots represent average expression in the tissue of individual patients. The line represents the mean, and error bars show SD. CRTAC1 is significantly downregulated in ILD but not COPD patients ( P ‐value < 0.0001) (one‐way ANOVA) (control donors n = 91, ILD n = 194, COPD n = 144). For each single‐cell cohort, the gene–gene correlations with CRTAC1 within the SFTPC+ AT‐2 cells were calculated. The indicated genes were selected based on their common direction of correlation across cohorts. The bar graph shows the gene categories most strongly correlated with CRTAC1 based on “UniProt keywords”. The dotted line marks a correlation coefficient of zero. The bar graph shows the gene categories most strongly correlated with CRTAC1 belonging to the GO category of “transcription regulators”. The dotted line marks a correlation coefficient of zero. The bar graph shows the top correlated transcriptional regulators, predicted by ingenuity pathway analysis (IPA) for the CRTAC1 gene–gene correlations. The dotted line marks a correlation coefficient of zero. Diffusion map of human AT2 cells colored by cell type identity and inferred pseudotime. The line plot illustrates smoothed expression levels of the indicated genes across the human AT2 pseudotime trajectory. Diffusion map of mouse AT2 cells colored by cell type identity and inferred pseudotime. The line plot illustrates smoothed expression levels of the indicated genes across the (Niu et al , ) mouse AT2 pseudotime trajectory. Immunofluorescence analysis of SPRR1A, KRT8 as well as SFTPC in IPF ( n = 3) and control samples ( n = 2). A high‐throughput experimental workflow for plasma proteomics (Niu et al , ) allowed for profiling of two independent cohorts of ILD patients (Munich, n = 30 and Hannover, n = 81; healthy age‐matched controls, n = 30). All proteins quantified in plasma are shown, ranked by their abundance measured by mass spectrometry (MS‐intensity). The indicated proteins from the plasma analysis were selected based on their common direction of correlation with patient lung function in two independent patient cohorts with distinct clinical characteristics. The heatmap shows the predicted relative contribution of lung cell types to the association of protein biomarker signatures in plasma with lung function (forced vital capacity—FVC). Patients were split in two groups, one with a mild decline in lung function [FVC 60–100%] and one with severe loss of lung function [FVC 20–60%] and compared to healthy age‐matched controls.

Article Snippet: In brief, after deparaffinization, rehydration, and heat‐mediated antigen retrieval with citrate buffer (10 mM, pH = 6.0), sections were blocked with 5% bovine serum albumin for 1 h at room temperature followed by overnight incubation with the following primary antibodies at 4°C: rabbit anti‐TNC (abcam, ab108930, 1:100), rabbit anti‐SPRR1A (abcam, ab125374, 1:2,000), rabbit anti‐SFTPC (Sigma‐Aldrich, HPA010928 1:150), rabbit anti‐SSTR2 (abcam, ab134152, 1:50), rabbit anti‐YAP (abcam, ab205270, 1:500), rat anti‐KRT8 (University of Iowa Hybridoma Bank, 1:200), mouse anti‐PDGFRβ (Origene, TA506230, 1:50), mouse anti‐SFTPC (Santa Cruz Biotechnologies, sc‐518029, 1:50), chicken anti‐Krt5 (BioLegend, Poly9059, 1:1,000), goat anti‐SFN (abcam, ab77187, 1:250), goat anti‐DES (Santa Cruz Biotechnologies, sc‐7559, 1:100), and goat anti‐CD45 (LifeSpan Biosciences, LS‑B14248, 1:300).

Techniques: Expressing, Diffusion-based Assay, Immunofluorescence, High Throughput Screening Assay, Mass Spectrometry, Biomarker Assay

Journal: bioRxiv

Article Title: PLZF is a new substrate of CRBN with thalidomide and 5-hydroxythalidomide

doi: 10.1101/2020.02.28.969071

Figure Lengend Snippet: a , Chemical structures of thalidomide, pomalidomide and lenalidomide. b , Schematic diagram of the thalidomide-dependent in vitro binding; assay between CRBN and substrates using AlphaScreen technology. c , Detection of luminescent signals of thalidomide-dependent interactions between bls-CRBN and FLAG-GST-IKZF1. Dose-dependent signals (DMSO, 2.5, 5, 10, 25, 50, or 100 μM thalidomide) was analysed with an in vitro binding assay using AlphaScreen technology. d , Results of in vitro high-throughput screening, targeting 1,118 human transcription factors. Green and red spots denote known neosubstrates and candidate clones, respectively. e , Confirmation of thalidomide-dependency on six hit proteins using an in vitro binding assay. Interaction between bls-CRBN and FLAG-GST-protein in the presence of DMSO or 50 μM thalidomide was detected using AlphaScreen technology. f , In vitro binding assay for thalidomide, pomalidomide, and lenalidomide. Interaction between bls-CRBN and FLAG-GST-PLZF in the presence of DMSO, (3.125, 6.25, 12.5, 25, 50, 100, or 200 μM) thalidomide, pomalidomide or lenalidomide was analysed using AlphaScreen technology. All relative AS (AlphaScreen) signals were expressed as relative luminescent signal with luminescent signal of DMSO as one, and error bars mean ± standard deviation (n=3).

Article Snippet: Anti-CRBN rabbit mAb (Cell Signaling Technology, #71810), anti-PLZF rabbit mAb (Cell Signaling Technology, #39784), anti-PLZF rabbit pAb (GeneTex, GTX111046), anti-SALL4 rabbit pAb (Abcam, ab29112), anti-SALL4 mouse mAb (Santa Cruz Biotechnology, sc-101147), anti-DDB1 mouse mAb (Santa Cruz Biotechnology, sc-376860), anti-CUL4 mouse mAb (Santa Cruz Biotechnology, sc-377188), anti-RBX1 mouse mAb (Santa Cruz Biotechnology, sc-393640), and anti-ubiquitin mouse mAb (P4D1, Cell Signaling Technology, #3936) were used as primary antibodies.

Techniques: In Vitro, Binding Assay, Amplified Luminescent Proximity Homogenous Assay, High Throughput Screening Assay, Clone Assay, Standard Deviation

Journal: bioRxiv

Article Title: PLZF is a new substrate of CRBN with thalidomide and 5-hydroxythalidomide

doi: 10.1101/2020.02.28.969071

Figure Lengend Snippet: a , Schematic diagram of thalidomide metabolites by CYPs. b , In vitro binding assay for thalidomide and 5-hydroxythalidomide. Interaction between bls-CRBN and FLAG-GST-IKZF1, -SALL4, -PLZF in the presence of DMSO, thalidomide or 5-hydroxythalidomide (3.125, 6.25, 12.5, 25, 50, 100, or 200 μM) was analysed using AlphaScreen technology. c , Immunoblot analysis of AGIA-PLZF, AGIA-SALL4, or AGIA-PLZF in FLAG-CRBN expressing CRBN -/- HEK293T cells treated with DMSO, thalidomide, or 5-hydroxythalidomide for 16 h. d , Immunoblot analysis of endogenous SALL4 or PLZF protein levels in HuH7 cells treated with DMSO, thalidomide, or 5-hydroxytahalidomide for 24 h. e , Immunoblot analysis of endogenous PLZF or IKZF1 protein levels in THP-1 cells treated with DMSO, thalidomide, or 5-hydroxytahalidomide for 24 h.

Article Snippet: Anti-CRBN rabbit mAb (Cell Signaling Technology, #71810), anti-PLZF rabbit mAb (Cell Signaling Technology, #39784), anti-PLZF rabbit pAb (GeneTex, GTX111046), anti-SALL4 rabbit pAb (Abcam, ab29112), anti-SALL4 mouse mAb (Santa Cruz Biotechnology, sc-101147), anti-DDB1 mouse mAb (Santa Cruz Biotechnology, sc-376860), anti-CUL4 mouse mAb (Santa Cruz Biotechnology, sc-377188), anti-RBX1 mouse mAb (Santa Cruz Biotechnology, sc-393640), and anti-ubiquitin mouse mAb (P4D1, Cell Signaling Technology, #3936) were used as primary antibodies.

Techniques: In Vitro, Binding Assay, Amplified Luminescent Proximity Homogenous Assay, Western Blot, Expressing

Journal: bioRxiv

Article Title: PLZF is a new substrate of CRBN with thalidomide and 5-hydroxythalidomide

doi: 10.1101/2020.02.28.969071

Figure Lengend Snippet: a , Immunoblot analysis of AGIA-PLZF protein levels in AGIA-PLZF and FLAG-CRBN expressing HEK293T cells treated with DMSO or lenalidomide in the presence of DMSO, MG132, or MLN4924 for 9 h. b , Immunoblot analysis of endogenous PLZF protein levels in HEK293T cells or CRBN -/- HEK293T cells treated with DMSO or lenalidomide for 24 h. c , Immunoprecipitation of FLAG-CRBN in FLAG-CRBN and AGIA-PLZF expressing HEK293T cells treated with DMSO or lenalidomide in the presence of DMSO or MG132 for 8 h. Components of CRL FLAG-CRBN and AGIA-PLZF were detected using each specific antibody, as indicated. d , Ubiquitination of AGIA-PLZF in AGIA-PLZF and FLAG-CRBN expressing CRBN -/- HEK293T cells treated with DMSO or thalidomide in the presence of DMSO or MG132 for 10 h. AGIA-PLZF was immunoprecipitated using anti-AGIA antibody and the polyubiquitin chain on AGIA-PLZF was analysed by immunoblot. e , In vitro binding and ubiquitination assay of AGIA-PLZF. Empty vector, AGIA-PLZF, or FLAG-CRBN expressing HEK293T cells were lysed and the lysates were mixed. The first immunoprecipitation with anti-AGIA or anti-FLAG antibodies was performed in the presence of DMSO or 200 μM lenalidomide. The purified AGIA-PLZF or CRL4 FLAG-CRBN complex, including AGIA-PLZF and FLAG-CRBN, was incubated with recombinant E1, E2, and HA-ubiquitin in the presence of DMSO or 200 μM lenalidomide, and the second immunoprecipitation was performed using anti-AGIA antibody. Ubiquitination of PLZF was analysed by immunoblot.

Article Snippet: Anti-CRBN rabbit mAb (Cell Signaling Technology, #71810), anti-PLZF rabbit mAb (Cell Signaling Technology, #39784), anti-PLZF rabbit pAb (GeneTex, GTX111046), anti-SALL4 rabbit pAb (Abcam, ab29112), anti-SALL4 mouse mAb (Santa Cruz Biotechnology, sc-101147), anti-DDB1 mouse mAb (Santa Cruz Biotechnology, sc-376860), anti-CUL4 mouse mAb (Santa Cruz Biotechnology, sc-377188), anti-RBX1 mouse mAb (Santa Cruz Biotechnology, sc-393640), and anti-ubiquitin mouse mAb (P4D1, Cell Signaling Technology, #3936) were used as primary antibodies.

Techniques: Western Blot, Expressing, Immunoprecipitation, Ubiquitin Proteomics, In Vitro, Binding Assay, Plasmid Preparation, Purification, Incubation, Recombinant

Journal: bioRxiv

Article Title: PLZF is a new substrate of CRBN with thalidomide and 5-hydroxythalidomide

doi: 10.1101/2020.02.28.969071

Figure Lengend Snippet: a , Schematic diagram of PLZF and truncated PLZFs. b , In vitro binding assay using truncated PLZF. Thalidomide-dependent interaction between bls-CRBN and FLAG-GST-PLZF-full length (FL) or truncated FLAG-GST-PLZF was analysed in the presence of DMSO or 50 μM thalidomide using AlphaScreen technology. c , Schematic diagram of swapped PLZF mutants. d , In vitro binding assay using swapped PLZF mutants was performed using the same procedure as in . e , Amino acid sequences of ZNF1 and ZNF3 in PLZF. f , In vitro binding assay using point mutants of PLZF was performed using the same procedure as in . g , Immunoblot analysis of AGIA-PLZF protein levels in FLAG-CRBN and PLZF-WT, PLZF-ZNF1-GA, PLZF-ZNF3-GA, or PLZF-ZNF1,3-GA expressing CRBN -/- HEK293T cells treated with DMSO or thalidomide for 16 h. All relative AS (AlphaScreen) signals were expressed as relative luminescent signal with luminescent signal of DMSO as one. Error bars mean ± standard deviation (n = 3) and P values were calculated by one-way ANOVA with Tukey’s post-hoc test (NS = Not Significant, and **** P < 0.0001).

Article Snippet: Anti-CRBN rabbit mAb (Cell Signaling Technology, #71810), anti-PLZF rabbit mAb (Cell Signaling Technology, #39784), anti-PLZF rabbit pAb (GeneTex, GTX111046), anti-SALL4 rabbit pAb (Abcam, ab29112), anti-SALL4 mouse mAb (Santa Cruz Biotechnology, sc-101147), anti-DDB1 mouse mAb (Santa Cruz Biotechnology, sc-376860), anti-CUL4 mouse mAb (Santa Cruz Biotechnology, sc-377188), anti-RBX1 mouse mAb (Santa Cruz Biotechnology, sc-393640), and anti-ubiquitin mouse mAb (P4D1, Cell Signaling Technology, #3936) were used as primary antibodies.

Techniques: In Vitro, Binding Assay, Amplified Luminescent Proximity Homogenous Assay, Western Blot, Expressing, Standard Deviation

Journal: bioRxiv

Article Title: PLZF is a new substrate of CRBN with thalidomide and 5-hydroxythalidomide

doi: 10.1101/2020.02.28.969071

Figure Lengend Snippet: a, Sall4, Plzf or Crbn mRNA expression in E4 chicken embryos was analysed by wholemount in situ hybridization. Left panel shows whole chicken embryo and right panel shows right forelimb bud. b , Immunoblot analysis of AGIA-GgPlzf in AGIA-GgPlzf expressing DF-1 cells transfected with shContol (shGFP) or shPlzf expression vector. c , Immunoblot analysis of Plzf from tissue of chicken forelimb or hindlimb bud. Endogenous Plzf protein expression was detected by immunoblot using chicken embryos infected with RCAN virus packaging shControl or shPlzf (forelimb shControl (n = 4), forelimb shPlzf (n = 4), hindlimb shControl (n = 2) or hindlimb shPlzf (n = 4)). d , Limb skeletal stained with Victoria blue. Skeletal patterning of forelimb and hindlimb in E6 chicken embryos infected RCAN virus packaging shControl (n = 10) or shPlzf (n = 9) were analysed by Victoria blue staining. h; humerus, r; radius, u; ulna, fe; femur, fi; fibula, t; tibia. e , Teratogenic phenotypes of chicken embryos in .

Article Snippet: Anti-CRBN rabbit mAb (Cell Signaling Technology, #71810), anti-PLZF rabbit mAb (Cell Signaling Technology, #39784), anti-PLZF rabbit pAb (GeneTex, GTX111046), anti-SALL4 rabbit pAb (Abcam, ab29112), anti-SALL4 mouse mAb (Santa Cruz Biotechnology, sc-101147), anti-DDB1 mouse mAb (Santa Cruz Biotechnology, sc-376860), anti-CUL4 mouse mAb (Santa Cruz Biotechnology, sc-377188), anti-RBX1 mouse mAb (Santa Cruz Biotechnology, sc-393640), and anti-ubiquitin mouse mAb (P4D1, Cell Signaling Technology, #3936) were used as primary antibodies.

Techniques: Expressing, In Situ Hybridization, Western Blot, Transfection, Plasmid Preparation, Infection, Virus, Staining