primary human airway epithelial (hae) cells  (MatTek)

Journal: Emerging Microbes & Infections

Article Title: Mutations in SARS-CoV-2 variant nsp6 enhance type-I interferon antagonism

doi: 10.1080/22221751.2023.2209208

Figure Lengend Snippet: ΔSGF alters host responses causing extensive cytokine expression. (A) Volcano plots from nCounter Analysis for mouse whole lung specimens and human airway epithelial cultures (HAE) at specified days post infection. All comparisons are between nsp6 mutant and WT. Horizontal dotted line corresponds to a p -value cutoff of 0.05 and vertical lines correspond to −0.6 and 0.6 log2(fold change). (B) Venn diagram of differentially expressed genes between four conditions (Day 2 mouse, Day 4 mouse, Day 6 mouse, and Day 2 HAE). (C) Bubble plot of the top 20 statistically significant canonical pathways by ascending p -value from comparison analysis in Ingenuity Pathway Analysis. Dot size corresponds to –log( p -value). Colour corresponds to activation z-score communicating the directionality (activation or inhibition) for that pathway. Grey indicates z-score values which could not be calculated. (D) Bubble plot of the top 20 statistically significant upstream regulators by ascending p -value from comparison analysis in Ingenuity Pathway Analysis.

Article Snippet: Primary human airway epithelial (HAE) cells and culture medium for HAE cells were purchased from MatTek Life Science (Ashland, MA, USA).

Techniques: Expressing, Infection, Mutagenesis, Comparison, Activation Assay, Inhibition

Journal: Nature

Article Title: Inhibition of calcium-triggered secretion by hydrocarbon-stapled peptides

doi: 10.1038/s41586-022-04543-1

Figure Lengend Snippet: a , The design of synthesized SP9 with biotin or CPPs conjugated to the N terminus and Cy3 to the C terminus, respectively. Biotin–SP9–Cy3 was bound to streptavidin-conjugated C2 or CRM197. b , Diagram of the analysis of cumulated Cy3 intensities within individual HAE cells. c , Confocal collapse (projected) images of fixed HAE cells that were treated with SP9–Cy3 or SP9–Cy3 conjugated to bacterial toxins (C2, CRM197) or CPPs. Scale bar, 10 µm. The experiment was independently repeated twice with air–liquid interface (ALI) cultures from different donors with similar results. d , Quantitative analysis of intracellular Cy3 fluorescence for each peptide in MUC5AC + HAE cells. Box plots and data points are shown for n cells (indicated below each box plot). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by post hoc Dunnett’s test; *** P < 0.0001. e , Schematic of the peptide application and sample collection from HAE cells maintained under ALI conditions. f , Representative western blot immunofluorescence images for MUC5AC on an apical surface of untreated HAE cells (control), HAE cells treated with 10 μM of SP9–Cy3, or 10 μM of either SP9–Cy3 or P9–Cy3 conjugated to CPPs for 30 min before stimulation. Basl., MUC5AC secretion during a 30 min period before stimulation (baseline). Exp., MUC5AC secreted within a 30 min experimental period with (IL-13 + ATP) or without (IL-13) stimulation of HAE cells with 100 µM ATP. Cells were treated with IL-13 to induce mucous metaplasia. All of the original blots are shown in Supplementary Fig. . g , h , The ratio of baseline to reference wash secretion (fold increase in baseline secretion over reference secretion) ( g ) and the ratio of experimental to baseline (fold increase in stimulated secretion over baseline secretion) ( h ) for each condition in f . The numbers below the box plots indicate n for each condition, representing individual ALI cultures derived from four donors for each condition. Statistical analysis was performed using two-way ANOVA followed by post hoc Dunnett’s test; * P = 0.013.

Article Snippet: Primary HAE cells from several donors were obtained from Promocell at passage 2 or isolated from fresh tissues that were obtained during tumour resections or lung transplantation with fully consent of patients (Ethics approval: ethics committee Medical School Hannover, project no. 2701-2015).

Techniques: Synthesized, Fluorescence, Western Blot, Immunofluorescence, Derivative Assay

Journal: Nature

Article Title: Inhibition of calcium-triggered secretion by hydrocarbon-stapled peptides

doi: 10.1038/s41586-022-04543-1

Figure Lengend Snippet: a , Representative confocal images (z-sections) of fixed HAE cells treated with SP9-Cy3 or SP9-Cy3 conjugated to CPPs or biotin. Biotin-SP9-Cy3 was bound to streptavidin-conjugated C2 or CRM197. The experiment was repeated twice with ALI cultures from different donors with similar results. Scale bar, 10 µm. b , The diagram illustrates the analysis of intracellular localization of MUC5AC, Cy3, and DAPI in airway secretory cells. Fluorescence intensities of DAPI, AlexaFluor 488 (MUC5AC) and Cy3 were analysed within individual MUC5AC + cells at each z-section, normalized and fluorescence intensity traces calculated along the basolateral to apical cell axis. c , Representative western blot immunofluorescence images for MUC5AC on apical surface of untreated HAE cells (control 1 and 2) or HAE cells treated with 100 μM SP9-Cy3, PEN-SP9-Cy3, TAT-SP9-Cy3, PEN-P9-Cy3, or TAT-P9-Cy3 for 24 h before stimulation. Wash represents MUC5AC accumulated during culture and before start of experiment. Baseline represents unstimulated MUC5AC secretion during a 15 min period after removal of accumulated MUC5AC and experimental represents MUC5AC secreted within 15 min of stimulation with (ATP) or without (no ATP) 100 µM ATP. Lysate represents MUC5AC within HAE cells at the end of the experiment. Cells were treated with IL-13 to induce mucous metaplasia. All original blots are shown in Supplementary Fig . d , Box plots and data points show the ratio of experimental / baseline secretion (fold increase of stimulated secretion over baseline secretion) following 24 h preincubation with 100 μM of the respective peptides. Numbers below box-plots indicate n for each condition, representing individual ALI cultures derived from 4 donors for each condition. * p = 0.046 for HAE cells treated with 100 μM PEN-SP9-Cy3, and p = 0.016 for HAE cells treated with 100 μM TAT-SP9-Cy3, assessed by two-way ANOVA followed by post-hoc Dunnett`s test.

Article Snippet: Primary HAE cells from several donors were obtained from Promocell at passage 2 or isolated from fresh tissues that were obtained during tumour resections or lung transplantation with fully consent of patients (Ethics approval: ethics committee Medical School Hannover, project no. 2701-2015).

Techniques: Fluorescence, Western Blot, Immunofluorescence, Derivative Assay

Journal: bioRxiv

Article Title: SARS-CoV-2 B.1.617.2 Delta variant replication, sensitivity to neutralising antibodies and vaccine breakthrough

doi: 10.1101/2021.05.08.443253

Figure Lengend Snippet: a-d . Live virus replication in CaLu-3 lung cells comparing B.1.1.7 with B.1.617.2. Calu-3 cells were; infected with variants at MOI 0.1. Cells and supernatants containing released virus were collected for RNA isolation, western blot and TCID 50 at 8, 24 and 48h postinfection. a. viral loads were measured by qPCR in cell lysates. b. viral protein levels were detected in cell lysates. c-d Live virus produced from infected Calu3 cells was collected and used to infect permissive Vero Ace2/TMPRSS2 cells to measure c . viral loads in Vero cells or d . to measure TCID 50 /ml. e-f. Virus replication kinetics in human airway epithelial (HAE) system with air liquid interface ALI, using two B.1.617.2 isolates and two B.1.1.7 isolates. g. Live virus r eplication in airway epithelial organoid cultures. Airway epithelial organoids were infected with SARS-CoV-2 variants B.1.1.7 and B.1.617.2 at MOI 1. Cells were lysed 24 and 48h post-infection and total RNA isolated. qPCR was used to determine copies of nucleoprotein gene in organoid cells and infectivity of cell free virus measured by infection of Vero AT2 cells. Data represent are representative of two independent experiments, h and i. western blots of pseudotyped virus (PV) virions (h) and cell lysates (i) of 293T producer cells following transfection with plasmids expressing lentiviral vectors and SARS-CoV-2 S B.1.617.1 and Delta variant B.1.617.2 versus WT (Wuhan-1 with D614G), probed with antibodies for HIV-1 p24 and SARS-Cov-2 S2. j. Single round infectivity on Calu-3 by spike B.1.617.2 and B.1.617.1 versus WT D614G parental plasmid PV produced in 293T cells. Data are representative of three independent experiments. k. Growth kinetics of B.1.617.1 and B.1.617.2 variants. Viral isolates of B.1.617.1 and B.1.617.2 [200 50% tissue culture infectious dose (TCID 50 )] were inoculated into Calu-3 cells and the copy number of viral RNA in the culture supernatant was quantified by real-time RT-PCR over time. TCID 50 of released virus in supernatant was also measured over time. Assays were performed in quadruplicate. *, P <0.05 by Mann-Whitney U test.. ns, non-significant; * p<0.05; ** p < 0.01. ***p<0.001, ****p<0.0001 (-) uninfected cells. Data are representative of two independent experiments

Article Snippet: Primary nasal human airway epithelial (HAE) cells at air-liquid interface (ALI) were purchased from Epithelix and the basal MucilAir medium (Epithelix) was changed every 2-3 days for maintenance of HAE cells.

Techniques: Infection, Isolation, Western Blot, Produced, Transfection, Expressing, Variant Assay, Plasmid Preparation, Quantitative RT-PCR, MANN-WHITNEY