Journal: The Journal of Biological Chemistry

Article Title: Neutrophilic Cathepsin C Is Maturated by a Multistep Proteolytic Process and Secreted by Activated Cells during Inflammatory Lung Diseases *

doi: 10.1074/jbc.M115.707109

Figure Lengend Snippet: Processing of proCatC in PLB-985 cells cultured in the presence or absence of synthetic inhibitors. A, cell surface CD11b expression was analyzed by flow cytometry. Undifferentiated PLB-985 were cultured for 1 week in the presence of ICatS (10 μm), ICatC (2 μm), or after adding the DMSO/DMF containing buffer alone. Cell lysates were analyzed by Western blotting using anti-CatC antibody (Ab1). The percentage of CatC, PR3, and CatG activity was determined using the respective selective substrates for each protease and is given in the box. B, PLB-985 cells were differentiated into neutrophil-like in the presence of DMF and with or without ICatS (10 μm) or ICatC (2 μm) or DMSO/DMF. Cell lysates were analyzed by Western blotting using anti-CatC antibody (Ab1). The % of CatC, PR3, and CatG activity measured using their respective selective substrates were shown in the box. Cell surface CD11b expression was analyzed by flow cytometry as in A. C, undifferentiated PLB-985 cells cultured in the presence of E64c (100 μm), E64d (100 μm), or DMSO alone for 48 h were lysed. Cell lysates and supernatants of PLB-985 cells were analyzed by Western blotting using anti-CatC antibody Ab1 (left and right panels, respectively). Similar results were observed in five independent experiments.

Article Snippet: E64c ((2 S ,3 S )- trans- epoxysuccinyl- l -leucylamido-3-methylbutane) and E64d (2 S ,3 S-trans -(ethoxycarbonyloxirane-2-carbonyl)- l -leucine-(3-methylbutyl) amide) were from Santa Cruz Biotechnology.

Techniques: Cell Culture, Expressing, Flow Cytometry, DMSO/DMF Sterilization, Western Blot, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Neutrophilic Cathepsin C Is Maturated by a Multistep Proteolytic Process and Secreted by Activated Cells during Inflammatory Lung Diseases *

doi: 10.1074/jbc.M115.707109

Figure Lengend Snippet: Processing of proCatC in HL-60 cells cultured with or without synthetic inhibitors. A, HL-60 cells were cultured for 1 week in medium containing ICatS (10 μm), ICatC (2 μm), ICatS/ICatC (10 μm/2 μm), DMF, or DMSO/DMF, and then total cell lysates were analyzed by Western blotting using anti-CatC antibody (Ab1). The percentages of residual CatC, PR3, and CatG activity toward their respective selective substrates were given in the box. B, HL-60 cells were cultured for 1 week in medium containing ICatS (10 μm), ICatC (2 μm), and ICatS/ICatC inhibitors (10 μm/2 μm), and their lysates were analyzed by Western blotting using anti-CatC antibody (Ab2). C, HL-60 cells were cultured for 48 h in medium containing ICatS (10 μm), E64c (100 μm), or E64d (100 μm), and then total cell extracts were analyzed by Western blotting using anti-CatC antibody (Ab1). Similar results were observed in five independent experiments. D, diagram summary showing the processing of proCatC in HL-60 cells cultured in the presence of ICatS or E64d. Arrows indicate the position of proteolytic cleavages in proCatC. CP, cysteine protease. E, partial sequence of the CatC propeptide (residues 1–65) showing the N-terminal proteolysis-sensitive extension (residues 1–24) and the structurally conserved sequence found in CatL-like cysteine proteases (residues 24–65). Model structure was obtained using cysteine peptidase C (PDB code 4HWY; Ref. 37) as a template.

Article Snippet: E64c ((2 S ,3 S )- trans- epoxysuccinyl- l -leucylamido-3-methylbutane) and E64d (2 S ,3 S-trans -(ethoxycarbonyloxirane-2-carbonyl)- l -leucine-(3-methylbutyl) amide) were from Santa Cruz Biotechnology.

Techniques: Cell Culture, DMSO/DMF Sterilization, Western Blot, Activity Assay, Sequencing

Journal: Nature Communications

Article Title: Integrin beta 1 facilitates non-enveloped hepatitis E virus cell entry through the recycling endosome

doi: 10.1038/s41467-025-61071-y

Figure Lengend Snippet: ( A ) S10-3 LAMP-1-GFP cells were inoculated with eHEV (MOI = 20 GE/cell) or nHEV (MOI = 30 GE/cell) for 6 h or 2 h on ice and incubated at 37 °C for 10 h or 7 h, respectively. Genomes (magenta) were detected by RNA-FISH using the ORF1 probe. Scale bar = 5 μm. ( B ) and ( C ) S10-3 cells were treated with cathepsin inhibitor E64 or DMSO and infected with ( B ) nHEV (MOI = 30 GE/cell, n = 7) or ( C ) eHEV (MOI = 20 GE/cell, n = 6). E64 was added with virus for 24 h (“during”), or 24 h post-infection and throughout the course of infection (“after”). Infectivity was assessed 5 days post-infection. ( D ) and ( E ) S10-3 cells were treated with E64 or DMSO and infected with ( D ) nHEV (MOI = 30 GE/cell, n = 9) or ( E ) eHEV (MOI = 20 GE/cell, n = 7). 8 h later, the inoculum was replaced with fresh media containing drugs. 24 h post-inoculation, HEV capsid were detected by staining and genomes as in ( A ) and quantified using CellProfiler. ( F ) Maximum projections of E64-treated S10-3 LAMP-1-GFP cells inoculated with nHEV (MOI = 30 GE/cell). 24 h later, capsids (magenta) and genomes (yellow) were detected as in ( C ). Representative of n = 6 microscope fields. ( G ) S10-3 LAMP-1-GFP cells were treated with E64 or DMSO 30 min prior to inoculation with nHEV (MOI = 30 GE/cell). After 8 h at 37 °C, inoculum was replaced with fresh media containing drugs. Genomes were detected and analysed as in ( A ). ( H ) Proposed working model on HEV cell entry. The interaction of nHEV with ITGB1 triggers internalisation through Rab11+ recycling endosomes, while eHEV is routed into Rab5a+ early endosomes. Both particles traffic through Rab7+ late endosomes and reach Lamp1+ lysosomes. The capsid and envelope are degraded by lysosomal cathepsins, allowing the release of viral genomes into the cytosol through an unknown penetration mechanism. This figure was created in BioRender. Dao Thi, V. (2025). https://BioRender.com/f6k0uqw . All replicates are from three independent experiments. Statistical analysis was performed by unpaired two-tailed Student’s t test ( A , D , E ) or one-way ANOVA ( B , C , G ). ****: p < 0.0001; ns, non-significant.

Article Snippet: Bafilomycin A (Sigma), Concanamycin A (Biomol) and E64 (Selleckchem, 25 μM) were used at the indicated concentrations.

Techniques: Incubation, Infection, Virus, Staining, Microscopy, Two Tailed Test

Journal: Journal of Medical Virology

Article Title: Native and activated antithrombin inhibits TMPRSS2 activity and SARS‐CoV‐2 infection

doi: 10.1002/jmv.28124

Figure Lengend Snippet: Antithrombin inhibits activity of cathepsin L, while moderately affecting cathepsin B. Recombinant cathepsin L (A) or isolated cathepsin B (B) were incubated with AT (Anbinex), small molecule TMPRSS2 inhibitor CM or small molecule cathepsin inhibitor E64‐d, 1 h before the addition of fluorogenic substrate Z‐L‐R‐AMC (for cathepsin L) or Z‐R‐R‐AMC (for cathepsin B). Data are shown as means ± SEM derived from n = 3 experiments performed in triplicates. AT, antithrombin; CM, camostat mesylate; SEM, standard error of the mean.

Article Snippet: For analysis of purified TMPRSS2, serially diluted AT, activated AT, CM, or E64‐d were mixed with 25 µl of 2 µg/ml recombinant TMPRSS2 in assay buffer (50 mM Tris‐HCl, 0.154 mM NaCl pH 8.0) for 10 min at 37°C, followed by addition of 50 µl of 20 µM BOC‐QAR‐AMC protease substrate.

Techniques: Activity Assay, Recombinant, Isolation, Incubation, Derivative Assay