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: bioRxiv

Article Title: OrgaMeas: A pipeline that integrates all the processes of organelle image analysis

doi: 10.1101/2024.04.15.589634

Figure Lengend Snippet: (A) Representative images of HeLa cells deprived of amino acids and FBS (ST: starvation) in the presence or absence of protease inhibitors (PI), 10 μg/mL pepstatin A, and 10 μg/mL E-64d, for 1 h. Total lysosomes and activated lysosomes were stained with the pH-insensitive probe LysoPrime Green (left six panels) and the pH-sensitive probe LysoTracker Red (right six panels), respectively. Scale bar, 20 µm. Lower panels are magnified images of the regions surrounded by yellow dotted lines in the respective upper panels. Representative data from three independent experiments are shown. (B, C) Quantification of the lysosomal area and average diameter of individual lysosomes per cell (B) and the intensity of LysoTracker Red signal per cell (C) using OrgaMeas. More than 100 cells were analyzed per experimental group. Data are presented as individual means. ***p < 0.001, Dunnett’s multiple comparison test (Ct vs ST or ST + PI) in (B), Tukey’s multiple comparison test (Ct vs ST, Ct vs ST + PI, ST vs ST + PI) in (C). Representative data from three independent experiments are shown.

Article Snippet: CCCP, pepstatin A, and E-64d were purchased from Fujifilm Wako Chemicals (Osaka, Japan).

Techniques: Staining, Comparison