Journal: Cell Reports

Article Title: Recurrent emergence of SARS-CoV-2 spike deletion H69/V70 and its role in the Alpha variant B.1.1.7

doi: 10.1016/j.celrep.2021.109292

Figure Lengend Snippet: The route of SARS-CoV-2 S-mediated virus entry in cell lines is not altered by ΔH69/V70 spike (A) Schematic illustrating spike in producer cells with CMK targeting and blocking furin cleavage (left panel). In target cells, camostat inhibits TMPRSS2 and, therefore, cell fusion at the plasma membrane, and E64D blocks cathepsins and targets endocytic viral entry (right panel). (B) Western blots show that CMK inhibits spike S1/S2 cleavage in producer cells transfected with the S ΔH69/V70 plasmid, and spikes with altered S1/S2 cleavage are incorporated into the virions. Antibodies against HIV-1 p24 and spike S2 were used with anti-GAPDH as a loading control. (C) The viruses produced from transfected HEK293T cells in the presence of CMK were used to transduce target cells. The luciferase reading is used as a surrogate for the spike infectivity bearing with various S2/FL ratios. The data shown are technical triplicates or quadruplicates, and statical analysis was done using an unpaired t test. (D) Comparison of the infectivity of spike with the PBCS deleted (ΔPBCS) with and without ΔH69/V70. The effect of ΔH69/V70 is independent of the PBCS. (E) ΔH69/V70 does not alter the virus entry route. S pseudotyped lentiviruses bearing WT S, ΔH69/V70 S, or VSV-G were used to transduce 293T-ACE2 or 293T-ACE2/TMPRSS2 cells in the presence of E64D or camostat at different drug concentrations. The cells were then harvested after 2 days and assayed for luciferase expression, which was then normalized against the non-drug control (set as 100%). The data shown are technical duplicates. The data are representative of at least two independent experiments.

Article Snippet: E64D and camostat experiments: ACE2 or ACE2 and TMPRSS2 transfected 293T cells were either E64D (Tocris) or camostat (Sigma-Aldrich) treated for 3 hours at each drug concentration before the addition of a comparable amount of input viruses pseudotyped with WT, H69/V70 deletion or VSV-G (approx.

Techniques: Virus, Blocking Assay, Clinical Proteomics, Membrane, Western Blot, Transfection, Plasmid Preparation, Control, Produced, Transduction, Luciferase, Infection, Comparison, Expressing

Journal: Journal of Virology

Article Title: Bovine coronavirus enters HRT-18 cells via membrane fusion and clathrin-mediated endocytosis in a low pH-, dynamin-, cholesterol-, microtubule-, Rab7-, and Rab11-dependent manner

doi: 10.1128/jvi.01274-25

Figure Lengend Snippet: BCoV entry into HRT-18 cells depends on cathepsin. ( A ) The maximum safe concentrations of E64d were determined using the CCK-8 assay. ( B ) Western blot analysis was used to evaluate the BCoV N protein expression levels, with grayscale analysis performed and presented as a bar graph. ( C ) RT-qPCR was performed to assess the BCoV gene copy numbers. ( D ) TCID 50 assay was used to measure the BCoV viral titers in the cell supernatant. ( E ) IFA was used to detect the number of BCoV-infected cells. Scale bar = 100 µm. ( F ) RT-qPCR was used to evaluate the effect of E64d on the viral entry. ( G ) RT-qPCR was used to evaluate the effect of E64d on BCoV attachment. Data are presented as the mean ± SD of three independent experiments (not significant, P > 0.05; * P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: The inhibitors used in this study included SSAA09E3 (Cat HY-138102, MedChemExpress), a novel inhibitor that blocks the fusion of the viral membrane with the host cell membrane; CPZ (Cat C0982, Sigma), a clathrin-mediated endocytosis inhibitor; nystatin (Cat 475914, Sigma), a caveolae inhibitor that acts as a sterol-binding agent disrupting caveolae; blebbistatin (Cat 203391, Sigma), an inhibitor of micropinocytosis; dynasore (Cat T1848, TargetMol), a dynamin inhibitor; MβCD (Cat T4072, TargetMol), a cholesterol depletion inhibitor; chloroquine (Cat S6999, Selleck) and NH 4 Cl (Cat A9434, Sigma), a potent inhibitor of V-ATPase and a specific inhibitor of acidification of endosomal vesicles; colchicine (Cat HY-16569, MedChemExpress), which inhibits the polymerization of tubulin; E64d (Cat S7393, Selleck), a cathepsin inhibitor; and camostat (Cat HY-13512, MedChemExpress), a TMPRSS2 inhibitor.

Techniques: CCK-8 Assay, Western Blot, Expressing, Quantitative RT-PCR, Infection

Journal: The FEBS journal

Article Title: Verapamil treatment induces cytoprotective autophagy by modulating cellular metabolism.

doi: 10.1111/febs.14064

Figure Lengend Snippet: Fig. 7: Verapamil induces autophagic flux Western blots for LC3 protein levels after treatment with increasing concentrations of Verapamil (Ver; 50, 100, 200 µM; 6 h) alone or in combination with lysosomal protease inhibitors – E64d/Pepstatin A (E/PepA; 10 µM; 6 h) (A-E). Actin or ERK2 were used as a loading control. Representative images of the Western blots are shown (upper panels) together with the quantification of LC3-II fold increase in comparison to control, untreated cells (lower panels). Each performed experiment is presented as an individual data point set with mean (±SEM), n= 3 to 6.

Article Snippet: Verapamil chloride (Sigma-Aldrich, V4629), E64d (LKT Laboratories, E0003), Pepstatin A (Calbiochem, 516481), Chloroquine (Sigma-Aldrich, C6620), Sodium oxamate (Sigma-Aldrich, O2751).

Techniques: Western Blot, Control, Comparison

Journal: iScience

Article Title: Dynamic association of the intramembrane proteases SPPL2a/b and their substrates with tetraspanin-enriched microdomains

doi: 10.1016/j.isci.2023.107819

Figure Lengend Snippet:

Article Snippet: E-64d , Enzo Lifesciences , BML-PI107-0001.

Techniques: Virus, Recombinant, Agarose Gel Electrophoresis, Protease Inhibitor, Modification, Western Blot, Transfection, Extraction, Bicinchoninic Acid Protein Assay, Plasmid Preparation, Software