Journal: Cell death and differentiation

Article Title: Caspases rather than calpains mediate remodelling of the fodrin skeleton during human placental trophoblast fusion.

doi: 10.1038/cdd.2009.133

Figure Lengend Snippet: Figure 4 Effects of calpain inhibitors, general caspase inhibitor or individual caspase inhibitors 3, 8 and 9 on a-fodrin fragmentation in BeWo cells and primary term trophoblasts. (a) Inhibitors of caspases 3, 8 and 9 (20 mM, DEVD, IETD and LEHD, respectively) attenuated the generation of the 120 kDa a-fodrin fragment in forskolin-treated (20 mM) BeWo cells after 48 h cultivation. The inhibitors of calpain – calpeptin and calpain inhibitor III (20 mM, 48 h) – did not affect forskolin-induced fragmentation of a-fodrin in BeWo cells. The general caspase inhibitor (20 mM, Q-VD-OPh) completely blocked a-fodrin fragmentation in forskolin-treated (20 mM, 48 h) BeWo cells. (b) Similar effects were observed in primary term trophoblasts after 48 h cultivation. (c) Efficiency of calpeptin and calpain inhibitor III was tested in ionomycin (10 mM, 2 h)-treated BeWo cells and HUVEC. Although a-fodrin fragmentation was weak and not effected by calpain inhibitors in ionomycin-treated BeWo cells, ionomycin-induced pronounced generation of the 150/145 kDa a-fodrin doublet in HUVEC, which was blocked in the presence of calpeptin and calpain inhibitor III. (d) Forskolin treatment (20 mM, 48 h) led to a slight increase of cleavage products of caspases 3, 8 and 9 (arrows) compared with vehicle control (DMSO, 0.2%). Staurosporine (2 mM, 2 h), but not ionomycin (10 mM, 1.5 h) strongly induced activation of caspases 3, 8 and 9 in BeWo cells. Nitrocellulose membranes were stained with Ponceau S solution to verify equal protein loading and blotting efficiency. Western blots are representative for three independent experiments

Article Snippet: On the next day, cells were preincubated for 30 min with calpeptin (Z-Leu-NLe-CHO, Calbiochem, Darmstadt, Germany), calpain inhibitor III (Z-Val-Phe-CHO, Calbiochem), caspase 3 inhibitor peptide (Z-DEVD-FMK, BD PharMingen, Franklin Lakes, NJ, USA), caspase 8 inhibitor peptide (Z-IETD-FMK, BD PharMingen), caspase 9 inhibitor peptide (Z-LEHD-FMK, R&D Systems, Minneapolis, MN, USA), general caspase inhibitor (Q-VD-OPh, R&D Systems) or negative control peptide (Z-FA-FMK, BD PharMingen) at a concentration of 20mM each.

Techniques: Control, Activation Assay, Staining, Western Blot

Journal: Cell death and differentiation

Article Title: Caspases rather than calpains mediate remodelling of the fodrin skeleton during human placental trophoblast fusion.

doi: 10.1038/cdd.2009.133

Figure Lengend Snippet: Figure 5 Effects of calpain and caspase inhibitors on bhCG secretion and syncytialization in BeWo cells and primary term trophoblasts. (a) Calpeptin and calpain inhibitor III (20 mM), as well as individual caspase inhibitors of caspase 8 (IETD, 20 mM), caspase 3 (DEVD, 20 mM), caspase 9 (LEHD, 20 mM) and the general caspase inhibitor (Q-VD-OPh, 20 mM) decreased forskolin (20 mM)-induced bhCG secretion in BeWo cells after 48 h. Secreted bhCG was normalized to total protein of treated cells. Secretion from BeWo cells treated with forskolin without inhibitor is given as 100%. (b) Inhibitors showed similar effects on bhCG secretion in primary term trophoblasts after 48 h. bhCG secretion from untreated primary term trophoblasts is given as 100%. (c) Syncytialization of forskolin (20 mM)-treated BeWo cells was not affected by calpain inhibitors (calpeptin and calpain inhibitor III, 20 mM) and inhibitors of caspases 3 (DEVD, 20 mM) and 9 (LEHD, 20 mM) after 48 h. Caspase 8 inhibitor (IETD, 20 mM) and the general caspase inhibitor Q-VD-OPh decreased syncytialization to 30.9 and 30.7%, respectively, compared with BeWo cells treated with forskolin alone (36.0 %). Data are presented as mean±S.D. from three independent experiments performed in triplicates. *Pp0.05, **Pp0.01, ***Pp0.001

Article Snippet: On the next day, cells were preincubated for 30 min with calpeptin (Z-Leu-NLe-CHO, Calbiochem, Darmstadt, Germany), calpain inhibitor III (Z-Val-Phe-CHO, Calbiochem), caspase 3 inhibitor peptide (Z-DEVD-FMK, BD PharMingen, Franklin Lakes, NJ, USA), caspase 8 inhibitor peptide (Z-IETD-FMK, BD PharMingen), caspase 9 inhibitor peptide (Z-LEHD-FMK, R&D Systems, Minneapolis, MN, USA), general caspase inhibitor (Q-VD-OPh, R&D Systems) or negative control peptide (Z-FA-FMK, BD PharMingen) at a concentration of 20mM each.

Techniques:

Journal: Biology of reproduction

Article Title: Immunolocalization of acidic and basic fibroblast growth factors in porcine uterine and conceptus tissues.

doi: 10.1095/biolreprod56.6.1527

Figure Lengend Snippet: FIG. 1. Western blot analysis of porcine uterine protein extract at Day 12 of gestation. a) Molecular weight markers (x 10 3) (lane 1), total CM-50 sephadex-extracted uterine protein (25 g, lanes 2, 4, and 6), human recombinant bFGF (100 ng, lanes 3 and 5). Lanes 1 and 2 were stained for total protein by colloidal gold stain. Lanes 3 and 4 were in- cubated with anti-bFGF antibody (0.15 Ixg/ml). Lanes 5 and 6 were in- cubated with rabbit IgG (0.15 .Lg/ml). b) Sephadex-extracted uterine pro- tein (25 jIg, stained for total protein by colloidal gold) in nonreducing 7.5-15% PAGE. Bovine aFGF (500 ng, lanes 2, 4, and 6) and sephadex extract (25 g, lanes 3, 5, and 7). Immunoreactive aFGF bands are ob- served in lanes 2 and 3 (incubated with anti-aFGF antibody). The absence of these bands is noted in lanes 4 and 5 (anti-aFGF antibody immunoab- sorbed with bovine aFGF) and in lanes 6 and 7 (incubated with rabbit IgG).

Article Snippet: For aFGE PAGE was performed using 7.5-15% acrylamide gel and bovine aFGF (cat. #132-FA; R&D Systems, Minneapolis, MN) as standard.

Techniques: Western Blot, Molecular Weight, Recombinant, Staining, Incubation

Journal: Computational and Structural Biotechnology Journal

Article Title: Potential Drug Discovery for COVID-19 Treatment Targeting Cathepsin L Using a Deep Learning-Based Strategy

doi: 10.1016/j.csbj.2022.05.023

Figure Lengend Snippet: Prediction dataset validation with a cell-free CTSL activity detection system. A, Among the available molecules, the top 50 molecules from the prediction dataset were chosen for verifying the inhibition effect against CTSL in a cell-free system at a single dose of 100 μM. Twelve of the 50 predicted molecules displayed over 50% inhibition against CTSL, and the top 5 were Mg-132, Z-FA-FMK, leupeptin hemisulfate, Mg-101 and calpeptin, with inhibition efficiencies greater than 90%. The data are expressed as the mean of three individual trials. B-F, Five predicted CTSL inhibitors, Mg-132(B), Z-FA-FMK(C), leupeptin hemisulfate(D), Mg-101(E) and calpeptin(F), with inhibition efficiencies greater than 90% at 100 μM were further tested for determination of the half maximal inhibitory concentration (IC 50 ) in the cell-free system. Corresponding molecular structure was drawn by Chemdraw. Non-linear fit to a variable response curve from one representative experiment with three replicates is shown (black lines). The data are expressed as the mean ± s.e.m.

Article Snippet: Mg-132 (Cat. No. S2619), Z-FA-FMK (Cat. No. S7391), leupeptin hemisulfate (Cat. No. S7380), Mg-101 (Cat. No. S7386), calpeptin (Cat. No. S7396), daptomycin (Cat. No. S1373), beta-lapachone (Cat. No. S7261) and other molecules and drugs were purchased from Selleck (Selleckchem, Houston, TX, USA).

Techniques: Activity Assay, Inhibition, Concentration Assay

Journal: Computational and Structural Biotechnology Journal

Article Title: Potential Drug Discovery for COVID-19 Treatment Targeting Cathepsin L Using a Deep Learning-Based Strategy

doi: 10.1016/j.csbj.2022.05.023

Figure Lengend Snippet: The predicted CTSL inhibitors from bioactive compounds prevent SARS-CoV-2 pseudovirus infection in Huh7 cells in vitro. A, Schematic of the predicted CTSL inhibitor assay setup. Huh7 cells were pretreated with different drugs 1 hour(h) before infection with SARS-CoV-2 pseudovirus or SARS-CoV-2 B.1.351 (Beta) variant pseudovirus at the same dose (1.3×10 4 TCID 50 /ml). Pseudovirus infection and cell viability were evaluated 24 h later by a luciferase activity and MTT assay, respectively. B-F, Inhibition of pseudovirus infection by different doses of Mg-132 (B), Z-FA-FMK (C), Leupeptin Hemisulfate (D), Mg-101 (E), and Calpeptin (F) and viability of Huh7 cells treated with different doses of the drugs as indicated. Non-linear fit to a variable response curve from one representative experiment with four replicates is shown (red lines). Cytotoxic effect on Huh7 cells exposed to increasing concentrations of drugs in the absence of virus is also shown (blue lines). The CC 50 , EC 50 , and SI values of this graph are indicated. n=4. The data are expressed as the mean ± s.e.m. G-K, Inhibition of SARS-CoV-2 B.1.351(Bata) variant pseudovirus infection by different doses of Mg-132 (G), Z-FA-FMK (H), Leupeptin Hemisulfate (I), Mg-101 (J), and Calpeptin (K) and viability of Huh7 cells treated with different doses of the drugs as indicated. Non-linear fit to a variable response curve from one representative experiment with four replicates is shown (purple lines). Cytotoxic effect on Huh7 cells exposed to increasing concentrations of drugs in the absence of virus is also shown (blue lines). The CC 50 , EC 50 , and SI values of this graph are indicated. CC 50 : 50% cytotoxic concentration. EC 50 : half maximal effective concentration. SI: the selectivity index, which is calculated as the ratio of CC 50 and EC 50 . n=4. The data are expressed as the mean ± s.e.m.

Article Snippet: Mg-132 (Cat. No. S2619), Z-FA-FMK (Cat. No. S7391), leupeptin hemisulfate (Cat. No. S7380), Mg-101 (Cat. No. S7386), calpeptin (Cat. No. S7396), daptomycin (Cat. No. S1373), beta-lapachone (Cat. No. S7261) and other molecules and drugs were purchased from Selleck (Selleckchem, Houston, TX, USA).

Techniques: Infection, In Vitro, Variant Assay, Luciferase, Activity Assay, MTT Assay, Inhibition, Concentration Assay

Journal: Computational and Structural Biotechnology Journal

Article Title: Potential Drug Discovery for COVID-19 Treatment Targeting Cathepsin L Using a Deep Learning-Based Strategy

doi: 10.1016/j.csbj.2022.05.023

Figure Lengend Snippet: The predicted CTSL inhibitors from bioactive compounds prevent live SARS-CoV-2 infection in Huh7 cells in vitro. A, Schematic of the predicted CTSL inhibitor assay setup. Huh7 cells were pretreated with different drugs 1 h before infection with live SARS-CoV-2 at the same dose (0.5 moi), followed by changing to fresh medium with the indicated concentrations of drugs 1 h later. The detection of infected cells was performed 48 h later by using an immunofluorescence assay. B-D, Inhibition of live SARS-CoV-2 infection by different doses of Mg-132 (B), Z-FA-FMK (C), and Leupeptin Hemisulfate (D). Non-linear fit to a variable response curve from one representative experiment with three replicates is shown (blue lines). The EC 50 value of this graph is indicated. n = 3. The data are expressed as the mean ± s.e.m.

Article Snippet: Mg-132 (Cat. No. S2619), Z-FA-FMK (Cat. No. S7391), leupeptin hemisulfate (Cat. No. S7380), Mg-101 (Cat. No. S7386), calpeptin (Cat. No. S7396), daptomycin (Cat. No. S1373), beta-lapachone (Cat. No. S7261) and other molecules and drugs were purchased from Selleck (Selleckchem, Houston, TX, USA).

Techniques: Infection, In Vitro, Immunofluorescence, Inhibition

Journal: Computational and Structural Biotechnology Journal

Article Title: Potential Drug Discovery for COVID-19 Treatment Targeting Cathepsin L Using a Deep Learning-Based Strategy

doi: 10.1016/j.csbj.2022.05.023

Figure Lengend Snippet: Molecular docking results of CTSL inhibitors in the crystal structure of human CTSL (5MQY). A-G, 3D structure of the human CTSL (5MQY) showing the main residues involved in the protein-ligand interaction of Compound 35 (A), Mg-132 (B), Z-FA-FMK (C), Leupeptin Hemisulfate (D), Calpeptin (E), Mg-101 (F) and Daptomycin (G). Compound 35 is a covalent inhibitor cocrystallized with human CTSL protein in 5MQY, used here as a positive control. Short intermolecular contacts with distances of < 4.0 Å between the ligand fragment (gray) and protein residues (dark green) are shown as dashed yellow lines. Structure visualization was by PyMol.

Article Snippet: Mg-132 (Cat. No. S2619), Z-FA-FMK (Cat. No. S7391), leupeptin hemisulfate (Cat. No. S7380), Mg-101 (Cat. No. S7386), calpeptin (Cat. No. S7396), daptomycin (Cat. No. S1373), beta-lapachone (Cat. No. S7261) and other molecules and drugs were purchased from Selleck (Selleckchem, Houston, TX, USA).

Techniques: Positive Control