Journal: Molecular Cell

Article Title: Human NLRP1 is a sensor of pathogenic coronavirus 3CL proteases in lung epithelial cells

doi: 10.1016/j.molcel.2022.04.033

Figure Lengend Snippet:

Article Snippet: A549 ACE2 & TMPRSS2 Cells , a549-hace2tpsa , Invivogen.

Techniques: Virus, Variant Assay, Clinical Proteomics, Recombinant, Microscopy, Plasmid Preparation, Mutagenesis, Construct, Software

Journal: Vaccines

Article Title: Deep Mutational Scanning to Predict Escape from Bebtelovimab in SARS-CoV-2 Omicron Subvariants.

doi: 10.3390/vaccines11030711

Figure Lengend Snippet: Figure 2. Bebtelovimab neutralization efficacy against the Omicron subvariants. (a) Location of mutations in the receptor-binding domain proteins of the Omicron subvariants: BA.2 and its three derivatives, BA.2.12.1, BA.2.75, XBB, and BA.4/5, and its two other derivatives, BA.4.6 and BQ.1.1. Conserved mutations among Omicron subvariants are highlighted in blue. Unique mutations in BA.2 derivatives and BA.4/5 derivatives are highlighted in green and red, respectively. Top number is the residue according to the spike protein of SARS-CoV-2 Wuhan-Hu-1. Data are adapted from Coronavirus Antiviral & Resistance Database of Stanford University “https://covdb.stanford.edu/ (accessed on 1 November 2022). (b) Neutralization efficacy of bebtelovimab in 293T/ACE2 cells. n = 4 technical replicates.

Article Snippet: Twenty-four hours after transfection, cells were incubated with human ACE2 (hACE2)-harboring green fluorescent protein (GFP) reporter viruses, which were generated by transfecting pcDNA4TO hACE2, psPAX2 (addgene #12260), and pLenti GFP into LentiX-293T cells with Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA).

Techniques: Neutralization, Binding Assay, Residue

Journal: bioRxiv

Article Title: ACE2 is necessary for SARS-CoV-2 infection and sensing by macrophages but not sufficient for productive viral replication

doi: 10.1101/2022.03.22.485248

Figure Lengend Snippet: A: HMDM and THP-1 cells were analysed by qPCR for ACE2 mRNA expression, with each data point showing an independent donor or experiment (n=3). B: HMDM were stimulated with IFNβ (10 ng/ml) for 6 h and protein extracts were analysed by immunoblot, alongside extracts from THP-1 cells (WT, THP-1-ACE2, THP-1-mSc). C: BAL macrophages from 3 donors were adhered overnight and lysed. Expression of ACE2 in BAL macrophages was analysed by immunoblot, relative to a loading control (Calnexin). Lysate from A549-cells overexpressing ACE2 were used as a positive control. D-E: Cells were infected with SARS-CoV-2 at MOI 0.5 or MOI 5. After 1h the virus inoculum was removed, cells were washed and cells or supernatants harvested at the indicated times. Cellular viral mRNA was analysed by qPCR (D-E), and infectious virions released into cell supernatants were measured by plaque assay (F-G). Data show the mean + SEM of 3-5 independent experiments, with data points representing individual experiments. Significance is indicated by asterisks: p ≤ 0.05 (*), p ≤ 0.001 (**), p ≤ 0.0001 (***) (two-way ANOVA, Tukey’s multiple comparison test).

Article Snippet: A lentiviral construct containing human ACE2 (Addgene 155295), or mScarlet (Addgene 85044) was cloned into pLV-CMV-MCS-IRES-Puro-Sin ( ) and packaged into lentivirus in HEK-293T cells by means of third generation lentiviral packaging plasmids ( ).

Techniques: Expressing, Western Blot, Control, Positive Control, Infection, Virus, Plaque Assay, Comparison

Journal: bioRxiv

Article Title: ACE2 is necessary for SARS-CoV-2 infection and sensing by macrophages but not sufficient for productive viral replication

doi: 10.1101/2022.03.22.485248

Figure Lengend Snippet: A: THP-1 cells were infected with SARS-CoV-2 (MOI 5), which was washed away after 1 h, and incubated for a further 72h, after which cell death was analysed by ATPlite assay. Data are presented as cell viability relative to mock, and are mean + SEM of 3 independent experiments. Significance is indicated by asterisks: p ≤ 0.05 (*), p ≤ 0.001 (**), p ≤ 0.0001 (***) (two-way ANOVA, Tukey’s multiple comparison test). B: Schematic of TBK1 (BX-795) inhibition. C-F: THP-1-ACE2 or Calu3 cells were stimulated with SARS-CoV-2 at MOI 5. After 1 h, the viral inoculum was removed and BX-795 added. Supernatants were harvested at 72 h and CXCL10 was analysed by ELISA (C) and viral titres were analysed by plaque assay (D,E). Data show mean + SEM of at least 3 independent experiments, with each individual data point representing a different experiment. Significance is indicated by asterisks: p ≤ 0.05 (*), p ≤ 0.001 (**), p ≤ 0.0001 (***) (C: one-way ANOVA, Tukey’s multiple comparison test; D,E: ratio-paired t-test).

Article Snippet: A lentiviral construct containing human ACE2 (Addgene 155295), or mScarlet (Addgene 85044) was cloned into pLV-CMV-MCS-IRES-Puro-Sin ( ) and packaged into lentivirus in HEK-293T cells by means of third generation lentiviral packaging plasmids ( ).

Techniques: Infection, Incubation, Comparison, Inhibition, Enzyme-linked Immunosorbent Assay, Plaque Assay