Journal: Scientific Reports

Article Title: Transcriptomics-based drug repositioning pipeline identifies therapeutic candidates for COVID-19

doi: 10.1038/s41598-021-91625-1

Figure Lengend Snippet: Haloperidol inhibits viral replication of SARS-CoV-2 in the Calu-3 lung cell line. ( A ) Calu-3 cells were infected with SARS-CoV-2 at an MOI of 0.05 for 72 h. Viral replication levels were determined by RT-qPCR from supernatant RNA using specific primers for the E gene. Viral RNA levels relative to DMSO are graphed. Error bars represent 3 or 4 independent experiments. One-way ANOVA analysis was used to determine significance. ( B ) Microscopy: Calu-3 cells were infected with SARS-CoV-2 at an MOI of 0.05 for 72 h. Cells were fixed with paraformaldehyde and used for immunofluorescence analysis with dsRNA antibody (SCICONS) and DAPI stain. Images were acquired and analyzed using ImageXpress Micro Confocal High-Content Imaging System.

Article Snippet: The inhibitory effects of haloperidol, clofazimine, valproic acid, and fluticasone were evaluated in SARS-CoV-2 infected Calu-3 cells (human lung epithelial cell line), with remdesivir also tested as a positive control.

Techniques: Infection, Quantitative RT-PCR, Microscopy, Immunofluorescence, Staining, Imaging

Journal: Cancer Research

Article Title: Epithelial to Mesenchymal Transition Is a Determinant of Sensitivity of Non–Small-Cell Lung Carcinoma Cell Lines and Xenografts to Epidermal Growth Factor Receptor Inhibition

doi: 10.1158/0008-5472.can-05-1058

Figure Lengend Snippet: Figure 3. A, NSCLC lines were grown as s.c. xenografts in nu/nu mice and dosed for 14 days with erlotinib (100 mg/kg qd) or vehicle control. Statistical evaluation of percent tumor growth inhibition by repeated measures ANOVA was done. H292, H358, and H441 were found to be sensitive to EGFR inhibition in vivo. No correlation between tumor growth rate and sensitivity to EGFR inhibition was observed. Solid bars, erlotinib dosing period. B, tumor cell lysates were evaluated for the expression of epithelial and mesenchymal proteins. Tumors were grown to a volume of f300 mm3, excised, and flash frozen in liquid nitrogen. Tumor tissues were pulverized while frozen, subjected to detergent lysis and SDS-PAGE as described, and immunoblots probed with antibodies to E-cadherin, h-catenin, Brk, fibronectin, and vimentin. Similar with in vitro results, E-cadherin expression was restricted to erlotinib-sensitive lines and vimentin to insensitive lines. Antibody to GAPDH was used as a loading control.

Article Snippet: The human NSCLC lines (H292, H358, H322, H441, A549, Calu6, H460, H1703, SW1573, Calu3, Colo699, H2122, A427, H1437, H1299, Hop92, and H23) were cultured in the appropriate American Type Culture Collection recommended supplemented media.

Techniques: Control, Inhibition, In Vivo, Expressing, Lysis, SDS Page, Western Blot, In Vitro

Journal: Molecular Cancer Therapeutics

Article Title: Targeted therapy of orthotopic human lung cancer by combined vascular endothelial growth factor and epidermal growth factor receptor signaling blockade

doi: 10.1158/1535-7163.mct-06-0416

Figure Lengend Snippet: Figure 2. Functional analysis of VEGFR2 and EGFR signaling and signaling blockade for human lung cancer and endothelial cells in vitro. A, Western blot analysis of VEGFR2 and EGFR activation for human NSCLC and endothelial cell lines. Human lung adenocarcinoma (PC14-PE6, H358, A427, H441, and Calu-3), human lung squamous (H226, SK-MES-1, and SW900), and endothelial (HUVEC, HPAEC, and MLEC) cell lysates were subjected to Western blot analyses with antibodies directed against pVEGFR2, pEGFR, or actin. Lane 1, PC14-PE6; lane 2, H358; lane 3, A427; lane 4, H441; lane 5, Calu-3; lane 6, H226; lane 7, SK-MES-1; lane 8, SW900; lane 9, HUVEC; lane 10, HPAEC; lane 11, MLEC. B, blockade of VEGFR2 and EGFR phosphorylation and resultant impairment of Akt and MAPK phosphorylation by ZD6474 treatment of MLEC and H441 lung adenocarcinoma cells. MLEC or H441 cells were treated with ZD6474 for 2 h then stimulated with VEGF165 (20 ng/mL) for 30 min or EGF (20 ng/mL) for 15 min and then lysed for Western blotting. C, effects of ZD6474 upon migration, invasion, and proliferation of H441 human lung cancer or endothelial cells. For the migration and invasion assays, HUVEC, HPAEC, MLEC, and H441 were treated with ZD6474 in serum-free medium containing 1% bovine serum albumin. For the proliferation assay, HUVEC, HPAEC, MLEC, and H441 were treated with ZD6474 in medium containing serum for 72 h and counted by the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide method. D, effects of ZD6474 upon apoptosis of H441 human lung cancer or endothelial cells. For apoptosis, MLEC and H441 cells were treated with ZD6474 in serum-free medium for 48 h in the presence or absence of EGF (20 ng/mL) or VEGF165 (20 ng/mL), and apoptosis was determined using the TUNEL assay and further evaluated by cleavage of caspase-3. Columns, mean percentage of total MLEC or H441 cells that are TUNEL or caspase-3 positive; bars, SD. One representative experiment of the three completed. * or #, P < 0.05; ** or ##, P < 0.001, versus control or VEGF/EGF alone, respectively.

Article Snippet: Cell Culture NCI-H441, NCI-H358, A427, and Calu-3 human lung adenocarcinoma cells and H226, SK-MES-1, and SW900 human squamous cell carcinoma cells were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Functional Assay, In Vitro, Western Blot, Activation Assay, Phospho-proteomics, Migration, Proliferation Assay, TUNEL Assay, Control

Journal: Molecular Cancer Therapeutics

Article Title: Targeted therapy of orthotopic human lung cancer by combined vascular endothelial growth factor and epidermal growth factor receptor signaling blockade

doi: 10.1158/1535-7163.mct-06-0416

Figure Lengend Snippet: Figure 4. Characterization of H441 human lung adenocarcinoma growth after orthotopic injection into the mouse lung. A, tumor growth of H441 in the lungs of mice. Logarithmically growing H441 cells in suspension (5 105 cells and 50 Ag growth factor–reduced Matrigel in 50 AL PBS) were injected into the left lungs of mice. Mice were killed on days 5, 10, and 15 following tumor injection, and primary lung and pleural tumor and pleural effusion were assessed. Columns, mean; bars, SD. *, P < 0.05, versus day 5. B, expression of VEGFR2, EGFR, and their ligands in H441 lung tumors. Primary lung tumors were fixed and stained with antibodies directed against VEGF, VEGFR2, pVEGFR2, EGF, TGFa, EGFR, and pEGFR. The reaction intensity of positive cells was measured in 10 random 0.039-mm2 fields at 200 magnification using Optimas Image Analysis software and was expressed as percentage of tumor cell expression to tumor-free tissue expression. Columns, mean; bars, SD. **, P < 0.001, versus normal lung. C, proliferation of and VEGFR2 and EGFR activation by tumor-associated endothelial cells in H441 lung tumors. Primary lung tumor tissues were frozen in optimal cutting temperature compound, cut, fixed, and stained with specific antibodies for pVEGFR2, pEGFR, or bromodeoxyuridine (BrdU; green fluorescence) and CD31 (red fluorescence). Overlay of the images was used to identify pVEGFR2, pEGFR, or bromodeoxyuridine-positive endothelial cells (yellow fluorescence). Bar, 100 Am. D, proliferation of tumor-associated endothelial cells in lung tumors. Bromodeoxyuridine-positive endothelial cells (yellow fluorescence) were counted in 10 random 0.039 mm2 fields at 200 magnification, and the percentage of positive cells was calculated. Columns, mean; bars, SD. **, P < 0.001, versus normal lung.

Article Snippet: Cell Culture NCI-H441, NCI-H358, A427, and Calu-3 human lung adenocarcinoma cells and H226, SK-MES-1, and SW900 human squamous cell carcinoma cells were obtained from the American Type Culture Collection (Manassas, VA).

Techniques: Injection, Suspension, Expressing, Staining, Software, Activation Assay, Fluorescence

Journal: Cell

Article Title: Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant

doi: 10.1016/j.cell.2020.09.032

Figure Lengend Snippet: SARS-CoV-2 D614G S Protein Variant Enhances Infectivity of Pseudotyped Lentiviruses in Cell Culture (A) Lentiviral virions bearing either GFP or Luciferase transgenes, and pseudotyped with either SARS-CoV-2 D614 or D614G S proteins, were produced by transfection of HEK293 cells and used to transduce human Calu3 lung cells, Caco2 colon cells, and either HEK293 or SupT1 cells stably expressing ACE2 and TMPRSS2. Relative infectivity of D614G versus D614, with D614 set at one, was determined based on flow cytometry for percent GFP positivity or on bulk luciferase activity. Each point represents the mean ± SD for transduction with lentiviral stocks derived from independent transfections, each value of which is the mean of three technical replicates. P values are the ratio paired t test (two-tailed). (B) Lentiviral virions bearing a luciferase transgene, pseudotyped with either SARS-CoV-2 D614 or D614G S proteins, were produced by transfection of HEK293 cells and used to transduce human HEK293 cells transiently transfected with plasmids encoding the indicated ACE2 orthologs. Relative infectivity of D614G versus D614, with D614 set at one, was determined based on bulk luciferase activity. Each point represents the mean ± SD after transduction by using lentiviral stock derived from an independent transfection, each of which is the mean of three technical replicates. P values are ratio paired t test (two-tailed).

Article Snippet: Calu3 Homo sapiens , male, lung adenocarcinoma cells (ATCC HTB-55) were maintained in EMEM supplemented with 10% FBS.

Techniques: Variant Assay, Infection, Cell Culture, Luciferase, Produced, Transfection, Transduction, Stable Transfection, Expressing, Flow Cytometry, Activity Assay, Derivative Assay, Two Tailed Test

Journal: Cell

Article Title: Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant

doi: 10.1016/j.cell.2020.09.032

Figure Lengend Snippet:

Article Snippet: Calu3 Homo sapiens , male, lung adenocarcinoma cells (ATCC HTB-55) were maintained in EMEM supplemented with 10% FBS.

Techniques: Binding Assay, Virus, Recombinant, Transfection, Luciferase, Bradford Protein Assay, Plasmid Preparation, Software, Single Particle, Microscopy