Journal: Nature Communications

Article Title: STAT2 signaling restricts viral dissemination but drives severe pneumonia in SARS-CoV-2 infected hamsters

doi: 10.1038/s41467-020-19684-y

Figure Lengend Snippet: a Schematic representation of SARS-CoV-2 inoculation schedule. WT (blue), STAT2 −/− (red), and IL28R-a −/− (purple) hamster strains were inoculated intranasally with 2 × 10 5 TCID 50 of passage 4 or 2 × 10 6 of passage 6 SARS-CoV-2. Outcomes derived from inoculation with passage 4 or passage 6 SARS-CoV-2 is designated by circles (P4, n = 3) or squares (P6, n = 4). On the indicated days post inoculation (p.i.), organs and blood were collected to determine viral RNA levels and infectious viral load. Viral loads in the indicated organs were quantified by RT-qPCR ( b , d – f ) or virus titration ( c ). b , e Viral RNA levels in the lungs (day 2 and day 3 p.i. of each genotype ( n = 3); day 4 p.i. of each genotype ( n = 7)) ( b ) or the indicated organs on day 4 p.i. ( n = 4 for each genotype) ( e ) were normalized against β-actin mRNA levels and transformed to estimate viral genome equivalents (vge) content per weight of the tissue (Fig. ). c Infectious viral loads in the lung on day 4 p.i. are expressed as the number of infectious virus particles per 100 mg of lung tissue ( n = 7 for each genotype). d Viral RNA levels in the blood (day 2 and day 3 p.i. of each genotype ( n = 3); day 4 p.i. of each genotype ( n = 7)) were calculated from a standard of infectious virus and expressed as TCID 50 equivalents per ml blood. Dotted lines indicate lower limit of quantification (LLOQ) or lower limit of detection (LLOD). f Viral RNA levels in hamsters after treatment with a previously described single-domain antibody. Hamsters were either left untreated (blue, n = 5) or treated with VHH-72-Fc (green, n = 4) and sacrificed on day 4 p.i. Viral RNA levels were determined in the lungs, normalized against β-actin, and fold changes were calculated using the 2 (−ΔΔCq) method compared to the mean of untreated control. g Inhibition of JAK/STAT signaling by Ruxolitinib can rescue SARS-CoV-2 virus replication in human airway epithelial cells from the antiviral effect of type I IFN. Calu-3 (human airway epithelial) cells were left untreated or treated with Ruxolitinib (4 µM), IFN-α (10 IU/ml), or a combination of both ( n = 8 for each condition). Treatment was initiated 4 h before infection and was continued through the whole experiment. Cultures were infected with P6 SARS-CoV-2 (MOI of 0.1), and 48 h p.i., cell culture supernatant was collected, RNA was extracted, and the amount of vRNA was quantified using RT-qPCR. A serial dilution of the same virus stock was used to generate a standard curve for absolute quantification. The data shown are mean ± SEM. Statistical significance between groups was calculated by Kruskal–Wallis with two-sided Dunn’s post hoc test ( b – d , g ) or by an unpaired two-sided t test ( f ). P values: * P = 0.010 ( c ), *** P = 0.0009 and * P = 0.02 ( d ), **** P < 0.0001 ( f ), * P = 0.022 and * P = 0.013 (left to right in g ); ns not significant.

Article Snippet: Calu-3 (human airway epithelial) cells were plated at 5 × 10 4 cells/well in a 96-well plate and incubated overnight with 4 µM of the JAK1/2 inhibitor Ruxolitinib (Toronto Research Chemicals, ON, Canada).

Techniques: Derivative Assay, Quantitative RT-PCR, Titration, Transformation Assay, Inhibition, Infection, Cell Culture, Serial Dilution

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: iScience

Article Title: The FDA-approved drug Auranofin has a dual inhibitory effect on SARS-CoV-2 entry and NF-κB signaling

doi: 10.1016/j.isci.2022.105066

Figure Lengend Snippet:

Article Snippet: Calu-3 cells (epithelial lung adenocarcinoma cells) , Elabscience , Cat#EP-CL-0054.

Techniques: Control, Virus, Recombinant, Bradford Assay, MTS Assay, Extraction, Lysis, Labeling, Amplex Red Cholesterol Assay, Plasmid Preparation, Software, Laser-Scanning Microscopy, Imaging, Microscopy, High Content Screening, Fluorescence

Journal: Frontiers in Medicine

Article Title: Cigarette Smoke Affects Dendritic Cell Populations, Epithelial Barrier Function, and the Immune Response to Viral Infection With H1N1

doi: 10.3389/fmed.2020.571003

Figure Lengend Snippet: (A–E) CS decreased viral replication in influenza H1N1-infected Calu-3 cells, whereas it was unchanged in PCLS. Calu-3 cells were exposed twice to either air or two doses of CS at the ALI. Following exposure, the cells were post-incubated for 24 h and then infected with influenza H1N1 (5,000 ffu/well for Calu-3 and 25,000 ffu/well for PCLS) at the apical surface. Reduction of TEER was observed after high dose CS or influenza H1N1 infection in exposed Calu-3 cells 48 h post infection. Virus titer was reduced in CS-exposed Calu-3 cells but not in CS-exposed PCLS. LDH release was reduced in CS-exposed and influenza H1N1-infected Calu-3 cells but not in PCLS. Every symbol represents an independent experiment (and donor for human PCLS). n = 4 independent experiments with four technical replicates each. * p < 0.05, ** p < 0.01, *** p < 0.001 compared with respective air control within uninfected/infected group analyzed by Tukey's multiple comparisons test, ## p < 0.01 uninfected vs. infected in air or CS exposed cells, analyzed by Tukey's multiple comparisons test.

Article Snippet: The TEER of the Calu-3 cells was measured using an EVOM2™ Epithelial Voltohmmeter (World Precision Instruments, Friedberg).

Techniques: Infection, Incubation, Virus

Journal: Frontiers in Medicine

Article Title: Cigarette Smoke Affects Dendritic Cell Populations, Epithelial Barrier Function, and the Immune Response to Viral Infection With H1N1

doi: 10.3389/fmed.2020.571003

Figure Lengend Snippet: CS exposure induced morphological changes in confluent Calu-3 cells due to cytotoxicity, which were enhanced by cytopatic effects of influenza H1N1 infection. Calu-3 cells were exposed to either air or two doses of CS at the ALI. Following exposure, the cells were post-incubated for 24 h and then infected with influenza H1N1 (5,000 ffu/well) at the apical surface. The inoculum was removed and cells were post-incubated for 48 h prior to the staining. Fixated Calu-3 cells were stained for pan cytokeratin (green) and cell nuclei with DAPI (blue) after (A) air only, (B) low dose CS, (C) high-dose CS, (D) air + influenza H1N1, (E) low dose CS + influenza H1N1, and (F) high-dose CS + influenza H1N1. Asterisk (*) shows cell detachment.

Article Snippet: The TEER of the Calu-3 cells was measured using an EVOM2™ Epithelial Voltohmmeter (World Precision Instruments, Friedberg).

Techniques: Infection, Incubation, Staining

Journal: Frontiers in Medicine

Article Title: Cigarette Smoke Affects Dendritic Cell Populations, Epithelial Barrier Function, and the Immune Response to Viral Infection With H1N1

doi: 10.3389/fmed.2020.571003

Figure Lengend Snippet: (A–F) CS impaired antiviral cytokine release upon H1N1 infection in Calu-3 cells and human PCLS. Calu-3 cells and human PCLS were exposed to either air or two doses of CS at the ALI. Following exposure, the cells/PCLS were post-incubated for 24 h and then infected with influenza H1N1 (5,000 ffu/well for Calu-3 and 25,000 ffu/well for PCLS) at the apical surface. Antiviral cytokines were induced in air-exposed and influenza H1N1-infected cells/PCLS but were inhibited in CS-exposed cells/PCLS 48 h post infection. Every symbol represents an independent experiment (and donor for human PCLS). n = 4 with four technical replicates, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared with respective air control within uninfected/infected group analyzed by Tukey's multiple comparisons test, ## p < 0.01, ### p < 0.001, #### p < 0.0001 air control vs. air infected control, analyzed by Tukey's multiple comparisons test.

Article Snippet: The TEER of the Calu-3 cells was measured using an EVOM2™ Epithelial Voltohmmeter (World Precision Instruments, Friedberg).

Techniques: Infection, Incubation

Journal: Frontiers in Medicine

Article Title: Cigarette Smoke Affects Dendritic Cell Populations, Epithelial Barrier Function, and the Immune Response to Viral Infection With H1N1

doi: 10.3389/fmed.2020.571003

Figure Lengend Snippet: (A–F) CS impaired pro-inflammatory immune response in influenza H1N1-infected Calu-3 cells and PCLS. Calu-3 cells and human PCLS were exposed to either air or two doses of CS at the ALI. Following exposure, the cells/PCLS were post-incubated for 24 h and then infected with influenza H1N1 (5,000 ffu/well for Calu-3 and 25,000 ffu/well for PCLS) at the apical surface. Pro-inflammatory cytokines were induced in air-exposed and influenza H1N1-infected cells/PCLS but were inhibited in CS-exposed cells/PCLS 48 h post infection. Every symbol represents an independent experiment (and donor for human PCLS). n = 4 with four technical replicates each, * p < 0.05, ** p < 0.01, *** p < 0.001 compared with respective air control within uninfected/infected group, ### p < 0.001 air control vs. air infected control, analyzed by Tukey's multiple comparisons test.

Article Snippet: The TEER of the Calu-3 cells was measured using an EVOM2™ Epithelial Voltohmmeter (World Precision Instruments, Friedberg).

Techniques: Infection, Incubation

Journal: Frontiers in Medicine

Article Title: Cigarette Smoke Affects Dendritic Cell Populations, Epithelial Barrier Function, and the Immune Response to Viral Infection With H1N1

doi: 10.3389/fmed.2020.571003

Figure Lengend Snippet: (A–F) CS impaired T cell activating response to influenza H1N1-infected Calu-3 cells and PCLS. ALI cultured Calu-3 cells and human PCLS were exposed to either air or CS (at low and high dose). Following exposure, the cells/PCLS were post-incubated for 24 h and then infected with influenza H1N1 (5,000 ffu/well for Calu-3 and 25,000 ffu/well for PCLS) at the apical surface. T cell activating cytokines were induced in air-exposed and influenza H1N1-infected cells/PCLS but were inhibited in CS-exposed cells/PCLS 48 h post infection. Every symbol represents an independent experiment (and donor for human PCLS). n = 4 with four technical replicates each, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared with respective air control within uninfected/infected group, # p < 0.05, ## p < 0.01 air control vs. air infected control, analyzed by Tukey's multiple comparisons test.

Article Snippet: The TEER of the Calu-3 cells was measured using an EVOM2™ Epithelial Voltohmmeter (World Precision Instruments, Friedberg).

Techniques: Infection, Cell Culture, Incubation

Journal: Emerging Microbes & Infections

Article Title: HA N193D substitution in the HPAI H5N1 virus alters receptor binding affinity and enhances virulence in mammalian hosts

doi: 10.1080/22221751.2024.2302854

Figure Lengend Snippet: Growth curves of the rCT/W811-HA 193N and rCT/W811-HA 193D viruses in avian- and mammalian-origin cells. Cell monolayers of DF-1 (A), CEF (B), MDCK (C), A549 (D), Calu-3 (E), and NHBE (F) were infected with the rCT/W811-HA 193N and rCT/W811-HA 193D viruses at an MOI of 0.01 for 72 h. TCID 50 virus titres were measured in the supernatants at the indicated time points. The data are presented as the mean values of three inoculated wells ± the SEM for each time point and are representative of three independent experiments. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates p < 0.0001.

Article Snippet: Human lung epithelial Calu-3 and A549 cells were purchased from Korean Cell Line Bank (KCLB) (Seoul, Korea, no. 30055 and 10185), grown and maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS, 100 IU/mL of penicillin, and 100 μg/mL of streptomycin.

Techniques: Infection, Virus