Journal: Nature Communications

Article Title: Broad-spectrum CRISPR-mediated inhibition of SARS-CoV-2 variants and endemic coronaviruses in vitro

doi: 10.1038/s41467-022-30546-7

Figure Lengend Snippet: a Scheme used for antiviral treatment of air–liquid-interface (ALI) cultures of human primary bronchial epithelial cells (hPBECs). hPBECs were transduced with NES-Cas13d and differentiated to ALI cultures. The ALI cultures were infected with 229E at an MOI of 0.05, with SARS-CoV-2 WA1 at an MOI of 0.6, or with Omicron at an MOI of 0.1. At 6 hpi, the culture was transfected with crRNA using LNP. At 48, 72 hpi, the apical surfaces of ALI cultures were washed with PBS and the wash solution was collected for measuring virus titer. b The titer of 229E virus released on the apical surface of ALI cultures was determined by RT-qPCR; n = 4, t = 3. c The titer of SARS-CoV-2 virus, including WA1 and Omicron strains, was determined at 48 hpi; n = 3, t = 3. n is the number of independent biological experiments. t is the number of technician replicates per biological replicate in the RT-qPCR assay. All source data in this figure are provided as a Source data file. P values are listed in supplementary Data , calculated by two-tailed Student’s t test.

Article Snippet: Human primary bronchial epithelial cells (hPBEC) were purchased from ATCC (PCS300010) and maintained in PneumaCult-Ex Plus Medium (StemCell, Canada) supplemented with hydrocortisone (0.5 μg/ml).

Techniques: Transduction, Infection, Transfection, Virus, Quantitative RT-PCR, Two Tailed Test

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Ezrin, a Membrane Cytoskeleton Cross-Linker Protein, as a Marker of Epithelial Damage in Asthma

doi: 10.1164/rccm.201802-0373OC

Figure Lengend Snippet: Ezrin depletion alters cell morphology and increases cellular permeability. (A) 16HBE cells were transfected with lentiviruses encoding for a control shRNA (Ctrl) or human ezrin-shRNA (EZR-shR1–3) tagged with GFP for 24 hours and then washed, and the cells were examined after a further 48 hours. GFP was detected by immunofluorescence (scale bar = 100 μm), quantitative RT-PCR, and Western blotting. ***P < 0.001 compared with the control group. (B) Phase-contrast images (original magnification, ×100 and ×400; scale bars = 50 μm) of 16HBE cells treated with control media (Ctrl), transfection control (Ctrl-shR), IL-13 (30 ng/ml), ezrin-shRNA (EZR-shR1–3), and IL-13 (30 ng/ml) + TG101348 (30 nM). White double-headed arrows show intercellular space enlargement; white single arrows show cellular protrusions. (C) Assessment of the permeability of the bronchial epithelium based on transepithelial electrical resistance (TER) (left panel) and fluorescein isothiocyanate (FITC)–dextran (right panel) in control media (Ctrl), transfection control (Ctrl-shR), IL-13 (30 ng/ml), ezrin-shRNA (EZR-shR2), and IL-13(30 ng/ml) + TG101348 (30 nM) groups. The data were analyzed using one-way ANOVA followed by Student-Newman-Keuls post hoc analysis. (C) Left panel: *P < 0.05 (IL-13 vs. control), #P < 0.05 (EZR-shR2 vs. Ctrl-shR); right panel: *P < 0.05 and **P < 0.01 compared with respective control subjects. Phase-contrast images are representative of those seen in three independent experiments.

Article Snippet: Cell Culture and Lentivirus shRNA Gene Transfection Human primary bronchial epithelial cells (PBECs; ScienCell Research Laboratories) and 16HBE cells were cultured as described previously ( 27 , 28 ).

Techniques: Permeability, Transfection, Control, shRNA, Immunofluorescence, Quantitative RT-PCR, Western Blot

Journal: Nucleic Acids Research

Article Title: Adaptive upregulation of DNA repair genes following benzo(a)pyrene diol epoxide protects against cell death at the expense of mutations

doi: 10.1093/nar/gkw873

Figure Lengend Snippet: BPDE induces expression of DNA repair genes in primary human cells, MCF7 cells and in buccal cells of smokers. ( A ) Induction of DDB2, XPC, XPF, XPG, POLH and p21 was analyzed in primary human bronchial epithelial cells (PBECs) 24 h upon exposure to BPDE (0.25 and 0.5 μM) by qRT-PCR. ( B ) To monitor the impact of pre-exposure on sensitivity to BPDE, PBECs were either not pre-exposed or pre-exposed to 0.25 μM BPDE and 24 h later exposed to 1 μM BPDE. Seventy-two hours later, cytotoxicity was detected by sub-G1 measurement. ( C ) MCF7 cells were exposed to 0.5 μM BPDE or 1 μM B(a)P. Twenty-four hours later, expression of 108 DNA repair genes was analyzed by qRT-PCR. Data for all genes showing an induction level above 2-fold is shown. For quantification, the expression was normalized to gapdh and ß-actin and the untreated control was set to 1. ( D ) qRT-PCR expression of DDB2, XPC and POLH in buccal cells of 12 male non-smokers (V1-V12) 24 h upon exposure to three cigarettes. For quantification, the expression was normalized to gapdh and ß-actin and the untreated control was set to 1.

Article Snippet: Human primary bronchial epithelial cells (PBECs) were purchased from Provitro (Berlin) and cultivated in Airway epithelial cell growth medium containing 10% fetal bovine serum.

Techniques: Expressing, Quantitative RT-PCR, Control