Journal: bioRxiv

Article Title: Cellular sialoglycans are differentially required for endosomal and cell-surface entry of SARS-CoV-2

doi: 10.1101/2024.06.24.600376

Figure Lengend Snippet: (A) Schematic showing cell-surface TMPRSS2-mediated CoV entry compared to endosomal cathepsin-mediated entry. Camostat mesylate inhibits the TMPRSS2 pathway, while E64d inhibits the endosomal pathway. (B-D) A549-D, A549-DT or Calu-3 cells (B) or A549-A or A549-AT cells (C-D) were pre-treated for 1 h at 37°C with DMSO, camostat (25 μM) or E64d (10 μM) diluted in media to the indicated concentrations, then infected with MERS-CoVpp (B) , SARS-CoV-2pp (C) or SARS-CoV-1-like pseudoparticles (D) for 2 h at 37°C. Inocula were removed and cells were incubated in complete media for 72 h, at which point luciferase activity was measured to assess viral entry. Data are expressed as percentage relative to DMSO control. Graphs show mean +/- SEM from at least three independent experiments performed in triplicate. Statistical significance was assessed by two-way ANOVA (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; ns, not significant).

Article Snippet: The TMPRSS2 inhibitor camostat mesylate was obtained from Sigma Aldrich (SML0057) and the cathepsin L inhibitor E64d was obtained from Tocris Bioscience (Cat# 4545).

Techniques: Infection, Incubation, Luciferase, Activity Assay, Control

Journal: bioRxiv

Article Title: Cellular sialoglycans are differentially required for endosomal and cell-surface entry of SARS-CoV-2

doi: 10.1101/2024.06.24.600376

Figure Lengend Snippet: (A) A549-A WT or CMAS KO cells were inoculated with the indicated pseudoparticles for 2 h at 37°, at which point the inocula were removed and cells were overlaid with complete media. After 72 h, lysates were collected to measure luciferase reporter activity to assess pseudoparticle entry. (B) A549-A WT or CMAS KO cells were pre-treated with DMSO vehicle or E64d (10 μM) for 1 h at 37°C, then inoculated with pseudoparticles as described above. Data are expressed as percentage relative to WT cells (A) or DMSO-treated control cells (B) . (A-B) Graphs show mean +/- SEM from at least three independent experiments performed in triplicate. Statistical significance was assessed by two-way ANOVA (***p<0.001; ****p<0.0001; ns, not significant).

Article Snippet: The TMPRSS2 inhibitor camostat mesylate was obtained from Sigma Aldrich (SML0057) and the cathepsin L inhibitor E64d was obtained from Tocris Bioscience (Cat# 4545).

Techniques: Luciferase, Activity Assay, Control

Journal: Cell Death & Disease

Article Title: FTY720 induces non-canonical phosphatidylserine externalization and cell death in acute myeloid leukemia

doi: 10.1038/s41419-019-2080-5

Figure Lengend Snippet: a MV4-11 cells were treated with 7.5 µM FTY720 or 1 μM RSL-3 (positive control) in the presence or absence of 2 µM Ferrostatin-1 (Fer-1) for 24 h, stained with APC-Ann V and 7-AAD and analyzed by flow cytometry. Mean ± SD, n = 3. Statistical significance for AnnV + 7AAD− (*) and AnnV + 7AAD+ (#) populations was determined by two-way ANOVA followed by Tukey’s multiple comparison test. **** p ≤ 0.0001; * p ≤ 0.05; #### p ≤ 0.0001; # p ≤ 0.05. b MV4-11 cells were treated with 7.5 µM FTY720 or 1.25 mM hydrogen peroxide (H 2 O 2 ; positive control) in the presence or absence of 5 mM N-acetyl-cysteine (NAC) for 22 h, stained with APC-Ann V and 7-AAD and analyzed by flow cytometry. Mean ± SD, n = 3 (DMSO, FTY720); n = 1 (H 2 O 2 ). Statistical significance for AnnV + 7AAD− (*) and AnnV + 7AAD+ (#) populations was determined by two-way ANOVA followed by Tukey’s multiple comparison test; **** p ≤ 0.0001; #### p ≤ 0.0001; ## p ≤ 0.01. c – d CSFE-labeled MV4-11 cells were treated with 7.5 μM FTY720 in the presence or absence of 2 µM Fer-1, 5 mM NAC or E64d/PepA (10 µg/mL each) in medium containing Ann V-AF594 ( c ) or YOYO3 ( d ). Images were obtained using the IncuCyte Live Cell Analysis System and quantified with the Basic Analyzer module. Mean ± SD, n = 3. Note: error bars are included for all data points but may be masked by symbols. c Percent of Ann V-AF594 positive CSFE-labeled cells versus time. d Percent of YOYO3 positive CSFE-labeled cells versus time. e CR-NT or ATG7-deficient MV4-11 cells were treated with 7.5 µM FTY720 or 250 nM ABT-199 (positive control) for 24 h, stained with APC-Ann V and 7-AAD and analyzed by flow cytometry. Mean ± SD, n = 3. Statistical significance for AnnV + 7AAD− (*) and AnnV + 7AAD+ (#) populations was determined by two-way ANOVA followed by Tukey’s multiple comparison test. ns, not significant; **** p ≤ 0.0001; #### p ≤ 0.0001; ## p ≤ 0.01; # p ≤ 0.05. Immunoblot included in inset. f MV4-11 cells were treated with 7.5 µM FTY720 in the presence or absence of E64d/PepA for 24 h and subjected to Ann V/7AAD flow cytometric analysis. Mean ± SD, n = 3. Statistical significance for AnnV + 7AAD− (*) and AnnV + 7AAD+ (#) populations was determined by two-way ANOVA followed by Tukey’s multiple comparison test. ns, not significant; **** p ≤ 0.0001; #### p ≤ 0.0001

Article Snippet: The following chemicals were purchased from the indicated sources: carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (z-VAD-fmk; #HY-16658) from MedChemExpress (Monmouth Junction, NJ, USA), E64d (#S7393) from Selleck Chemicals (Houston, TX, USA), pepstatin A (#260-085) and dynasore (#270-502) from Enzo Life Sciences, Inc. (Farmingdale, NY, USA), and Bafilomycin A1 (#AAJ61835MCR) from Thermo Fisher Scientific.

Techniques: Positive Control, Staining, Flow Cytometry, Comparison, Labeling, Cell Analysis, Western Blot