e 64d  (MedChemExpress)

Journal: PLOS Pathogens

Article Title: The impact of clade B lineage 5 MERS coronaviruses spike mutations from 2015 to 2023 on virus entry and replication competence

doi: 10.1371/journal.ppat.1013336

Figure Lengend Snippet: Treatment of cells with camostat targeting the cell surface TMPRSS2 pathway and E64d targeting the endocytic cathepsin L pathway was used to block infectious virus entry via each pathway. Experiments with infectious virus (A) or pseudovirus (B) is shown for the different spike variants in VeroE6/TMPRSS2 cells. (A) Relative viral load was measured the RNA copies of MERS-CoV using the UpE assay in the cell supernatant at 48 hpi. (B) Relative cell entry was measured by RLU. (A-B) 2015/GD01 was the reference for comparison. Figures show the representative data from 2 independent experiments each with two biological replicates for each experiment each drug dose. An inhibition curve was fitted using the Hill equation with inhibition in Graphpad Prism. (C) RT-qPCR to measure RNA expression of DPP4, TMPRSS2 and CTSL gene among difference cell lines. Expressions were normalized by GAPDH gene for each cell line and relative fold changes were compared to 293T cells as reference. Flow cytometry analysis of surface expression of DPP4 and TMPRSS2 among different cell lines. Background levels were determined by isotype controls as indicated in grey. (D) Caco2 cells representing the TMPRSS2 entry pathway model and Huh7 cells representing the endosomal entry pathway model were pretreated with Camostat (25 μM), E64d (10 μM) or in both. Pseudoviruses were infected on drug treated cells in quadruplicate wells. Data shown are a representative of two independent repeats.

Article Snippet: 3193) and E64d (Tocris, Cat. No. 4545) were serially diluted with 2% FBS DMEM medium and incubated on to VeroE6/TMPRSS2 cells for 2 hours prior infection.

Techniques: Blocking Assay, Virus, Comparison, Inhibition, Quantitative RT-PCR, RNA Expression, Flow Cytometry, Expressing, Infection