e-64d (#e8640)  (Merck & Co)

Journal: Nature

Article Title: PLSCR1 is a cell-autonomous defence factor against SARS-CoV-2 infection

doi: 10.1038/s41586-023-06322-y

Figure Lengend Snippet: a , Virus entry into Huh7.5 cells of the indicated genotypes. Cells were challenged with an HIV-1-based luciferase-expressing vector pseudotyped with SARS-CoV-2 spike (USA-WA1/2020) and assayed for luciferase activity at 48 hpi. ( n = 3). b , Virus entry into Huh7.5 cells inoculated with pseudovirus bearing spike proteins from SARS-CoV-2 Delta (left) or Omicron (right) ( n = 3). c , Viral entry efficiency into Huh7.5 cells inoculated with PsVs bearing fusion proteins from SARS-CoV ( n = 3), MERS-CoV ( n = 3), bat CoV-WIV1 ( n = 5) or VSV ( n = 3). d , Quantification of MHV-A59-GFP fluorescent intensity in LET1-ACE2 cells (MOI = 0.1, 24 hpi, n = 5). e , Levels of ACE2 in NC or PLSCR1 -KO A549-ACE2 cells using surface biotinylation. Membrane-impermeable biotin was used to label cell-surface proteins, followed by streptavidin pulldown. Surface ACE2 was then quantified by western blot. GAPDH was used as a cytosol marker. Bio-PD, biotin pulldown. f , Virus binding and internalization in NC or PLSCR1 -KO Huh7.5 cells. The amount of viral RNA in NC cells was normalized to 1 ( n = 4). g , Cleavage of the SARS-CoV-2 spike protein in NC or PLSCR1 -KO (PKO) A549-ACE2 cells infected with PsV carrying SARS-CoV-2 spike for the indicated time periods. Cells treated with E-64d (20 μM) served as a negative control ( n = 3). IB, immunoblot; NT, N-terminus; FP, fusion peptide; CT, C-terminus. h , Virus–cell fusion assay in Huh7.5 (middle) and 293T-ACE2 (right) cells ( n = 3). Fusion was measured by complemented (NanoLuc) activity. i , Left, representative images showing syncytia formation after co-culturing Huh7.5 cells of the indicated genotypes and 293T cells expressing SARS-CoV-2 spike and EGFP. Right, quantification of fusion activity in Huh7.5 cells of the indicated genotypes. Cell–cell fusion was measured by complemented NanoLuc activity ( n = 5). j , Left, representative images showing the formation of dsRNA foci in A549-ACE2 cells at 3 hpi. (MOI = 5). dsRNA was detected with a monoclonal rJ2 antibody (MABE1134). Right, percentage of cells with dsRNA foci. n = 10 image fields (NC, 209 cells; PLSCR1 -KO, 210 cells analysed). Data are mean ± s.d. P values from two-sided Student’s t -test in f , g , h (middle) and i (−Spike group), two-sided Student’s t -test with Welch’s correction in i (+Spike group), one-way ANOVA followed by Tukey’s multiple comparison test in a – d , h (right) and two-sided Mann–Whitney test in j . Scale bars, 200 μm ( i ) and 20 μm ( j ). All experiments performed three times, except a , g , i (four times).

Article Snippet: Camostat mesylate (SML0057), E-64d (E8640), brefeldin A (B652), hydroxychloroquine sulfate (H0915), cellulose (435244), poly- l -lysine hydrobromide (P9155) and polybrene (TR-1003) were obtained from Sigma.

Techniques: Luciferase, Expressing, Plasmid Preparation, Activity Assay, Western Blot, Marker, Binding Assay, Infection, Negative Control, Cell Fusion Assay, MANN-WHITNEY

Journal: Nature

Article Title: PLSCR1 is a cell-autonomous defence factor against SARS-CoV-2 infection

doi: 10.1038/s41586-023-06322-y

Figure Lengend Snippet: a , b , Quantification of viral entry efficiency in Huh7.5 cells inoculated with pseudoviruses bearing fusion proteins from HCoV-229E ( n = 5), HCoV-OC43 ( n = 3), hCoV-NL63 ( n = 3), hCoV-HKU1 ( n = 3), EBoV (n = 3) and HCV ( n = 5). EBoV: Ebola virus, HCV: Hepatitis C virus. c , Left, relative amount of intracellular viral RNA in Huh7.5 cells infected with DENV (Dengue virus type I) at an MOI = 0.5 for 24 h. The amount of viral RNA in NC cells was normalized to 1. ( n = 4) Right: Quantification of % infected cells in HeLa cells infected with HSV-1 VP26-GFP at an MOI of 0.1 for 48 h. d , Quantification of the relative entry efficiency of the indicated pseudovirus in Huh7.5 cells overexpressing (OE) PLSCR1 or IFITM3. The luminescence intensity in vector group was normalized to 1. n = 4. e , Schematic showing the dissection of the cell entry route of SARS-CoV-2. f , g , Effect of the indicated compounds on SARS-CoV-2 entry in Huh7.5 cells (MOI = 1, 48 hpi, n = 3) ( f ) and A549-ACE2 cells (MOI = 0.2, 24 hpi, n = 3) ( g ). E-64d: 20 μM, Camostat: 30 μM, Bfa (Brefeldin a): 10 μM, HCQ: 10 μM. Cells were treated with indicated compounds 2 h before infection. h , Quantification of SARS-CoV-2 infection in E-64d (20 μM) treated or untreated Huh7.5 cells overexpressing vector or PLSCR1 with or without ectopic expression of TMPRSS2 (MOI = 1, 48 hpi). ( n = 4) i , Left, dose response of indicated compounds on SARS-CoV-2 infection in Calu-3 (MOI = 1, 24 hpi, n = 4). The amount of viral RNA in DMSO group was normalized to 1. E64-d and Camostat groups share the same DMSO control group. Right, quantification of SARS-CoV-2 infection in Control or PLSCR1 -KO Calu-3 (MOI = 1, 24 hpi, n = 4) treated with indicated compounds (E-64d: 20 μM, Camostat: 20 μM). Cells were treated with the indicated compounds 2 h before infection. The amount of viral RNA in NC-DMSO group was normalized to 1. Data are mean ± s.d. P values were calculated using one-way ANOVA followed by Tukey’s multiple comparison test in a – c , d (HCoV-NL63 group), f , g , Brown–Forsythe and Welch ANOVA with Dunnett’s post-hoc test in d (SARS-CoV-2 and EBoV group), two-sided Student’s t -test in h , two-way ANOVA followed by Tukey’s multiple comparison test in i (left) or two-way ANOVA followed by Šídák’s multiple comparisons test in i (right). Experiments in this figure were performed three times.

Article Snippet: Camostat mesylate (SML0057), E-64d (E8640), brefeldin A (B652), hydroxychloroquine sulfate (H0915), cellulose (435244), poly- l -lysine hydrobromide (P9155) and polybrene (TR-1003) were obtained from Sigma.

Techniques: Infection, Plasmid Preparation, Dissection, Expressing

Journal: Nature

Article Title: PLSCR1 is a cell-autonomous defence factor against SARS-CoV-2 infection

doi: 10.1038/s41586-023-06322-y

Figure Lengend Snippet: a , Representative images showing the localization of indicated proteins in uninfected or infected WT A549-ACE2 cells treated with 20 μM E-64d at MOI = 20 for 4 h (left). Quantification of the average amount of spike and nucleocapsid double-positive particles per cell (right). n = 10 image fields (NC: 146 cells, KO: 131 cells). b , Representative images showing the localization of indicated proteins in uninfected or infected WT A549-ACE2 cells treated with 20 μM HCQ at MOI = 20 for 4 h (left). Quantification of the average amount of spike and nucleocapsid double-positive particles per cell (right). n = 10 image fields (NC: 141 cells, KO: 156 cells). c , Quantification of fluorescence intensities of Lysosensor in control or PLSCR1 -KO A549-ACE2 cells in the presence or absence of HCQ (20 μM). ( n = 3). d , Western blot showing the protein expression levels in 293T-ACE2 cells. Related to Fig. . e , Quantification of cell–cell fusion by co-culture of Huh7.5 cells overexpressing vector or PLSCR1 and 293T cells expressing SARS-CoV-2 spike. ( n = 6). f , Left, representative images showing the of distribution of SARS-CoV-2 spike and nucleocapsid protein in control or PLSCR1 -KO A549-ACE2 cells (MOI = 10, 4 hpi). Orange stars represent cells with dispersed and bright nucleocapsid signal. Blue stars represent cells with endosomal nucleocapsid signal. Right, quantification of the percentage of cells with dispersed or endosomal nucleocapsid signal. Number of cells analysed within each of 10–11 fields (left to right):142, 181, 146, 131, 141 and 156. n values are labelled on graph. Data are mean ± s.d. P values were calculated using two-sided Student’s t -test in a , b , f , two-sided Student’s t -test with Welch’s correction in e or one-way ANOVA followed by Tukey’s multiple comparison test in c . Scale bar in a , b , f : 20 μm, inlays: 5 μm. Experiments in this figure were performed three times.

Article Snippet: Camostat mesylate (SML0057), E-64d (E8640), brefeldin A (B652), hydroxychloroquine sulfate (H0915), cellulose (435244), poly- l -lysine hydrobromide (P9155) and polybrene (TR-1003) were obtained from Sigma.

Techniques: Infection, Fluorescence, Western Blot, Expressing, Co-Culture Assay, Plasmid Preparation

Journal: Frontiers in Immunology

Article Title: Tryptase Regulates the Epigenetic Modification of Core Histones in Mast Cell Leukemia Cells

doi: 10.3389/fimmu.2021.804408

Figure Lengend Snippet: Treatment of mast cell leukemia cells with tryptase inhibitor abrogates H3 cleavage in response to cytotoxic agents. (A) HMC-1 cells (0.5 x 10 6 cells/ml) were incubated with protease inhibitors: Pefabloc SC (Pefa; 0.1 mM), nafamostat (Nafa; 20 μM), E-64d (15 μM), Pepstatin A (Pep A; 50 μM) or EDTA (20 μM) for 30 min prior to treating the cells with LLME (400 μM) for 24 h. Subsequently, samples corresponding to equal numbers of cells were subjected to Western blot analysis for histone 3 (H3), with ß-actin as loading control. (B) Quantification of the signal intensity for cleaved H3, as % of total H3. The presented data are representative of at least two independent experiments, and are given as mean values ± SEM (n=3). ****p ≤ 0.0001. ns, not significant.

Article Snippet: Nafamostat mesylate (cat no N0289), staurosporine (cat no S4400), mefloquine (cat no M2319), Pefabloc SC (cat no 11429868001), pepstatin A (cat no 516481) and E-64d (cat no E8640) were from Sigma-Aldrich (Steinheim, Germany).

Techniques: Incubation, Western Blot