Journal: Journal of medical virology

Article Title: The impact of cycling hypoxia on the phenotype of HPV-positive cervical cancer cells.

doi: 10.1002/jmv.29280

Figure Lengend Snippet: FIGURE 5 The levels of mature cathepsins and their enzymatic activities are reduced under chronic hypoxia and cycling hypoxia (cycH) but do not affect apoptosis induction by Cisplatin (CDDP). (A) Immunoblot analyses of Cathepsin B (CTSB) and Cathepsin L (CTSL) levels in SiHa and HeLa cell lysates cultivated under 21% O2, 5.5% O2, 1% O2, or cycH for 24 h. Pre, precursor; int, intermediate; mat, mature cathepsin forms. Vinculin, representative loading control. (B) Enzymatic activities of CTSB and CTSL in SiHa cells after 24 h under the indicated O2 conditions. Normoxic cells treated with protease inhibitors (PI) served as negative control. Data are represented as mean enzymatic activities relative to the enzyme activity at 5.5% O2 ± SDs (n = 3). Asterisks indicate statistical significance (**p < 0.01; ***p < 0.001). (C) Comparative immunoblot analyses of mature CTSL and CTSB protein levels in total cell lysates (left panels) and cytosolic fractions (right panels) of SiHa and HeLa cells upon treatment for 24 h with 30 µM CDDP (SiHa) or 15 µM CDDP (HeLa) under the indicated O2 conditions. The lysosomal membrane marker LAMP1 indicates that the cytosolic fraction is efficiently depleted of lysosomes. n.d., not detected. Vinculin, representative loading controls. Glyceraldehyde 3‐phosphate dehydrogenase (GAPDH), loading control for HeLa total cell lysate. (D) SiHa and HeLa cells cultivated under 21% O2, 5.5% O2, 1% O2 or cycH were pretreated for 4 h with 10 µM E64, 1 µM CA‐074‐Me, or dimethyl sulfoxide (DMSO) as solvent control (−) before being exposed for 24 h to 30 µM CDDP (SiHa) or 15 µM CDDP (HeLa). Levels of poly(ADP‐ribose) polymerase (PARP) and cleaved (cl.) PARP were analyzed by immunoblot. α‐Tubulin, representative loading control.

Article Snippet: The following chemicals were used for treatment: Cisplatin (CDDP; Sigma‐Aldrich); Etoposide (Enzo Life Sciences); E64 (Cayman Chemical); CA‐074‐Me (Selleckchem); Z‐VAD(OMe)‐FMK (TargetMol).

Techniques: Western Blot, Control, Negative Control, Activity Assay, Membrane, Marker, Solvent

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Host Plants Indirectly Influence Plant Virus Transmission by Altering Gut Cysteine Protease Activity of Aphid Vectors *

doi: 10.1074/mcp.M116.063495

Figure Lengend Snippet: CathB enzymatic activity assays on turnip-reared (T-Myzus) and physalis-reared (P-Myzus) Myzus persicae, before and after treatment with the cysteine protease inhibitor E-64.

Article Snippet: Cathepsin Inhibition and PLRV Transmission Assays Adult aphids were placed in dishes covered by a Parafilm® sandwich membrane containing an aphid diet prepared with balanced amounts of amino acids ( 45 ) for 48 h. The adults were then removed, and first instar nymphs were transferred to a fresh diet containing the cysteine protease inhibitor E-64 (AG Scientific) at 0, 10, 30, or 50 μ m , for a 48h AAP.

Techniques: Activity Assay, Protease Inhibitor

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Host Plants Indirectly Influence Plant Virus Transmission by Altering Gut Cysteine Protease Activity of Aphid Vectors *

doi: 10.1074/mcp.M116.063495

Figure Lengend Snippet: Cathepsin inhibition modulates PLRV transmission by Myzus persicae. Proportion of plants infected with PLRV by aphids reared on turnip (T-Myzus) A) and on physalis (P-Myzus) B) after feeding on different concentrations of the cysteine protease inhibitor E-64.

Article Snippet: Cathepsin Inhibition and PLRV Transmission Assays Adult aphids were placed in dishes covered by a Parafilm® sandwich membrane containing an aphid diet prepared with balanced amounts of amino acids ( 45 ) for 48 h. The adults were then removed, and first instar nymphs were transferred to a fresh diet containing the cysteine protease inhibitor E-64 (AG Scientific) at 0, 10, 30, or 50 μ m , for a 48h AAP.

Techniques: Inhibition, Transmission Assay, Infection, Protease Inhibitor

Journal: Cell Host & Microbe

Article Title: A Replication-Competent Vesicular Stomatitis Virus for Studies of SARS-CoV-2 Spike-Mediated Cell Entry and Its Inhibition

doi: 10.1016/j.chom.2020.06.020

Figure Lengend Snippet: rVSV-SARS-CoV-2 S Infection Requires the Activity of Cysteine Cathepsin Proteases (A) Huh7.5.1 cells pre-treated for 1 h at 37°C with the indicated concentrations of NH 4 Cl were infected with pre-titrated amounts of rVSVs bearing SARS-CoV-2 S or EBOV GP. Infection was scored by eGFP expression at 16–18 h post-infection (average ± SD, n = 8 from 2 independent experiments). (B) Vero cells pre-treated for 90 min at 37°C with the indicated concentrations of pan-cysteine cathepsin inhibitor E-64 were infected with pre-titrated amounts of rVSVs bearing SARS-CoV-2 S, EBOV GP, or VSV G and scored for infection as above (average ± SD, n = 6 from 3 independent experiments, except n = 4 from 2 independent experiments for EBOV GP). (C) Vero cells pre-treated for 90 min at 37°C with the indicated concentrations of cathepsin L/B inhibitor FYdmk were infected with pre-titrated amounts of rVSVs bearing SARS-CoV-2 S, EBOV GP, or VSV G. Infection was scored as above (average ± SD, n = 6 from 3 independent experiments). (D) Vero cells and Vero cells overexpressing TMPRSS2 pre-treated for 120min at 37C with the indicated concentrations of camostat ]were infected with pre-titrated amounts of rVSVs bearing SARS-CoV-2 S and subsequently scored for infection. In panels (B)–(D), all comparisons were made between vehicle- and inhibitor-treated samples. ns,not statistically significant. ∗ p < 0.033, ∗∗∗ p < 0.001.

Article Snippet: Cysteine cathepsin inhibitor E-64 , Wako Chemicals , Cat# 331-40963.

Techniques: Infection, Activity Assay, Expressing