Journal: Biomedicines

Article Title: The Effects of Cathepsin B Inhibition in the Face of Diffuse Traumatic Brain Injury and Secondary Intracranial Pressure Elevation

doi: 10.3390/biomedicines12071612

Figure Lengend Snippet: Schematic representation of the experimental procedures utilized in the current study. ( A ) The top panel represents the experimental design for animals processed for molecular studies and ( B ) the lower panel depicts the experimental design for animals processed for histological studies. All animals in both groups had whisker nuisance task (WNT, yellow arrow) behavioral assessments carried out prior to injury and at 2w (13 d) post-injury. On the day of injury, day 0, animals either sustained a central fluid percussion injury (CFPI) or a sham injury (red arrowhead). From 15 min to 1 h following sham or CFPI, animals either had intracranial pressure (ICP) monitoring or ICP elevation to 20 mmHg. An initial bolus of either the cathepsin B inhibitor CA-074Me or 10% DMSO vehicle followed by implantation of an osmotic pump for continuous intracerebroventricular (ICV) administration was completed 1 h to 2 h post-injury followed by recovery from anesthesia. At 2w post-sham or CFPI, animals used for histological analysis were anaesthetized for bilateral ICV infusion of the cell-impermeable 10 kDa Alexa-Fluor-488 fluorescently tagged dextran (green arrow), followed by transcardial perfusion 1 h later.

Article Snippet: The solid form of the Cath B inhibitor CA-074 methyl ester (CA-074Me; MedChemExpress, Cat#: HY-100350, Monmouth Junction, NJ, USA) was stored at −20 °C until reconstitution in 100% dimethylsulfoxide (DMSO) at a concentration of 100 µg/µL.

Techniques: Whisker Assay

Journal: Biomedicines

Article Title: The Effects of Cathepsin B Inhibition in the Face of Diffuse Traumatic Brain Injury and Secondary Intracranial Pressure Elevation

doi: 10.3390/biomedicines12071612

Figure Lengend Snippet: Cathepsin B (Cath B) activity was reduced following 2w of the Cath B inhibitor CA-074Me infusion into the left lateral ventricle. Cath B activity was significantly decreased in the left and right lateral neocortex following CA-074Me infusion (filled boxes) compared to 10% DMSO control (vehicle; unfilled boxes). Box and whisker graph depicting the average fluorescent intensity indicating Cath B activity in the right and left side of the lateral neocortex and the liver of sham-injured control animals (n = 12; grey unfilled boxes for n = 6 saline-treated animals and filled boxes for n = 6 CA-074Me-treated animals), animals sustaining a traumatic brain injury (TBI) only (n = 13; light blue unfilled boxes for n = 6 saline-treated animals and filled boxes for n = 7 CA-074Me-treated animals), or animals sustaining a TBI followed by secondary intracranial pressure (ICP) elevation (n = 12; dark blue unfilled boxes for n = 6 saline-treated animals and filled boxes for n = 6 CA-074Me-treated animals). Note that Cath B activity in the liver was significantly higher than that in the cortex regardless of injury group. Additionally, ICP infusion of CA-074Me did not impact Cath B activity in the liver; however, CA-074Me infusion did significantly lower Cath B activity in the left and right cortex compared to DMSO controls. * p < 0.05 compared to injury type-matched vehicle control, # p < 0.05 compared to injury type-matched liver. Mean ± S.E.M.

Article Snippet: The solid form of the Cath B inhibitor CA-074 methyl ester (CA-074Me; MedChemExpress, Cat#: HY-100350, Monmouth Junction, NJ, USA) was stored at −20 °C until reconstitution in 100% dimethylsulfoxide (DMSO) at a concentration of 100 µg/µL.

Techniques: Activity Assay, Control, Whisker Assay, Saline

Journal: Biomedicines

Article Title: The Effects of Cathepsin B Inhibition in the Face of Diffuse Traumatic Brain Injury and Secondary Intracranial Pressure Elevation

doi: 10.3390/biomedicines12071612

Figure Lengend Snippet: Cathepsin B (Cath B) protein levels were slightly increased in animals infused with the Cath B inhibitor CA-074Me. ( A ) Representative chemiluminescent blot image of the two mature Cath B bands at 24/27 kDa, which was normalized to ( B ) total protein. Box and whisker graphs depicting average (n = 6/group) quantities of ( C ) total Cath B protein, ( D ) the upper band of the Cath B doublet, and ( E ) the lower band of the Cath B doublet for sham-injured animals (grey boxes), animals sustaining a central fluid percussion injury (CFPI; light blue boxes), and animals sustaining both CFPI and secondary intracranial pressure (ICP) elevation (dark blue boxes) followed by a 2w intracerebroventricular infusion of either 10% DMSO (vehicle, left half of graphs) or the Cath B inhibitor, CA-074Me (right half of graphs). Amount of Cath B for each case was calculated as a percentage compared to a consistent naïve control that was run on all membranes. While there were no significant differences found for total Cath B protein or the upper band of the observed doublet, there was a significant increase in Cath B levels of the lower band of the doublet in animals infused with CA-074Me compared to their vehicle counterparts. * p < 0.05 compared to vehicle. Mean ± S.E.M.

Article Snippet: The solid form of the Cath B inhibitor CA-074 methyl ester (CA-074Me; MedChemExpress, Cat#: HY-100350, Monmouth Junction, NJ, USA) was stored at −20 °C until reconstitution in 100% dimethylsulfoxide (DMSO) at a concentration of 100 µg/µL.

Techniques: Whisker Assay, Control

Journal: Biomedicines

Article Title: The Effects of Cathepsin B Inhibition in the Face of Diffuse Traumatic Brain Injury and Secondary Intracranial Pressure Elevation

doi: 10.3390/biomedicines12071612

Figure Lengend Snippet: Protein levels of Bcl-XL were unchanged regardless of injury or infusion group. ( A ) Representative chemiluminescent blot image of Bcl-XL at 30 kDa, which was normalized to ( B ) total protein. ( C ) Box and whisker graph depicting average (n = 6/group) quantities of Bcl-XL protein for sham-injured animals (grey boxes), animals sustaining a central fluid percusion injury (CFPI; (light blue boxes), and animals sustaining both CFPI and secondary intracranial pressure (ICP) elevation (dark blue boxes) followed by a 2w ICF infusion of either 10% DMSO (vehicle, left half of graph) or the Cath B inhibitor, CA-074Me (right half of graph). Amount of Bcl-XL for each case was calculated as a percentage compared to a consistent naïve control that was run on all membranes. Mean ± S.E.M.

Article Snippet: The solid form of the Cath B inhibitor CA-074 methyl ester (CA-074Me; MedChemExpress, Cat#: HY-100350, Monmouth Junction, NJ, USA) was stored at −20 °C until reconstitution in 100% dimethylsulfoxide (DMSO) at a concentration of 100 µg/µL.

Techniques: Whisker Assay, Control

Journal: Biomedicines

Article Title: The Effects of Cathepsin B Inhibition in the Face of Diffuse Traumatic Brain Injury and Secondary Intracranial Pressure Elevation

doi: 10.3390/biomedicines12071612

Figure Lengend Snippet: Protein quantification of BAK revealed no differences in the protein quantity regardless of group. ( A ) Representative chemiluminescent blot image of BAK at 25 kDa, which was normalized to ( B ) total protein. ( C ) Box and whisker graph depicting average (n = 6/group) quantities of BAK protein for sham-injured animals (grey boxes), animals sustaining a central fluid percussion injury (CFPI; light blue boxes), and animals sustaining both CFPI and secondary intracranial pressure (ICP) elevation (dark blue boxes) followed by a 2w ICF infusion of either 10% DMSO (vehicle, left half of graph) or the Cath B inhibitor, CA-074Me (right half of graph). Amount of BAK for each case was calculated as a percentage compared to a consistent naïve control that was run on all membranes. Mean ± S.E.M.

Article Snippet: The solid form of the Cath B inhibitor CA-074 methyl ester (CA-074Me; MedChemExpress, Cat#: HY-100350, Monmouth Junction, NJ, USA) was stored at −20 °C until reconstitution in 100% dimethylsulfoxide (DMSO) at a concentration of 100 µg/µL.

Techniques: Whisker Assay, Control

Journal: Biomedicines

Article Title: The Effects of Cathepsin B Inhibition in the Face of Diffuse Traumatic Brain Injury and Secondary Intracranial Pressure Elevation

doi: 10.3390/biomedicines12071612

Figure Lengend Snippet: Protein quantification of AIF revealed no differences in the protein quantity regardless of group. ( A ) Representative chemiluminescent blot image of the two observed AIF bands at ~67/72 kDa, which was normalized to ( B ) total protein. Box and whisker graphs depicting average (n = 6/group) quantities of ( C ) the upper band of the AIF doublet and ( D ) the lower band of the AIF doublet for sham-injured animals (grey boxes), animals sustaining a central fluid percussion injury (CFPI; light blue boxes), and animals sustaining both CFPI and secondary intracranial pressure (ICP) elevation (dark blue boxes) followed by a 2w intracerebroventricular infusion of either 10% DMSO (vehicle, left half of graphs) or the Cath B inhibitor, CA-074Me (right half of graphs). Amount of AIF for each case was calculated as a percentage compared to a consistent naïve control that was run on all membranes. Mean ± S.E.M.

Article Snippet: The solid form of the Cath B inhibitor CA-074 methyl ester (CA-074Me; MedChemExpress, Cat#: HY-100350, Monmouth Junction, NJ, USA) was stored at −20 °C until reconstitution in 100% dimethylsulfoxide (DMSO) at a concentration of 100 µg/µL.

Techniques: Whisker Assay, Control

Journal: Biomedicines

Article Title: The Effects of Cathepsin B Inhibition in the Face of Diffuse Traumatic Brain Injury and Secondary Intracranial Pressure Elevation

doi: 10.3390/biomedicines12071612

Figure Lengend Snippet: Representative fluorescent micrographs of membrane disruption in sham-injured animals (sham, ( A , D )), animals sustaining a traumatic brain injury (TBI, ( B , E )), and animals sustaining a TBI followed by secondary intracranial pressure (ICP) elevation (TBI+ 20 mmHg ICP elevation, ( C , F )) paired with 2w of intracerebroventricular infusion of 10% DMSO (vehicle ( A – C )) or the Cathepsin B inhibitor, CA-074Me ( D – F ). The left panel in blue depicts NeuroTrace Nissl-stained cells and the middle panel in green depicts cells containing a cell-impermeable Alexa-Fluor-488-tagged dextran (AF-dextran-488). The right panel is the overlay of the NeuroTrace and membrane-disrupted dextran images. The arrow heads indicate representative membrane-disrupted neurons. Scale bar 20 μm.

Article Snippet: The solid form of the Cath B inhibitor CA-074 methyl ester (CA-074Me; MedChemExpress, Cat#: HY-100350, Monmouth Junction, NJ, USA) was stored at −20 °C until reconstitution in 100% dimethylsulfoxide (DMSO) at a concentration of 100 µg/µL.

Techniques: Membrane, Disruption, Staining

Journal: Biomedicines

Article Title: The Effects of Cathepsin B Inhibition in the Face of Diffuse Traumatic Brain Injury and Secondary Intracranial Pressure Elevation

doi: 10.3390/biomedicines12071612

Figure Lengend Snippet: The total number of cells in the lateral neocortex layers V and VI is unaffected across injury and infusion groups. Bar graph depicting the average number of fluorescent NeuroTrace Nissl-stained neurons in sham-injured animals (grey boxes), animals sustaining a CFPI (light blue boxes), and animals sustaining both CFPI and secondary ICP elevation (dark blue boxes) followed by a 2w ICV infusion of either 10% DMSO (vehicle, left half of graphs) or the Cath B inhibitor, CA-074Me (right half of graphs). The mean number of cells was quantified per unit area (0.098 mm 2 ) and averaged for each animal (n = 6/group). Mean ± S.E.M.

Article Snippet: The solid form of the Cath B inhibitor CA-074 methyl ester (CA-074Me; MedChemExpress, Cat#: HY-100350, Monmouth Junction, NJ, USA) was stored at −20 °C until reconstitution in 100% dimethylsulfoxide (DMSO) at a concentration of 100 µg/µL.

Techniques: Staining

Journal: Biomedicines

Article Title: The Effects of Cathepsin B Inhibition in the Face of Diffuse Traumatic Brain Injury and Secondary Intracranial Pressure Elevation

doi: 10.3390/biomedicines12071612

Figure Lengend Snippet: There were no significant changes in membrane disruption regardless of injury or Cathepsin B inhibitor infusion. Bar graph highlighting a consistently low average (n = 6/group) percentage of membrane-disrupted neurons in sham-injured animals (grey boxes), animals sustaining a central fluid percussion injury (CFPI; light blue boxes), and animals sustaining both CFPI and secondary intracranial pressure (ICP) elevation (dark blue boxes) followed by a 2w ICV infusion of either 10% DMSO (vehicle, left half of graph) or the Cathepsin B inhibitor CA-074Me (right half of graph). Mean ± S.E.M.

Article Snippet: The solid form of the Cath B inhibitor CA-074 methyl ester (CA-074Me; MedChemExpress, Cat#: HY-100350, Monmouth Junction, NJ, USA) was stored at −20 °C until reconstitution in 100% dimethylsulfoxide (DMSO) at a concentration of 100 µg/µL.

Techniques: Membrane, Disruption

Journal: Biomedicines

Article Title: The Effects of Cathepsin B Inhibition in the Face of Diffuse Traumatic Brain Injury and Secondary Intracranial Pressure Elevation

doi: 10.3390/biomedicines12071612

Figure Lengend Snippet: Representative fluorescent micrographs of cathepsin B (Cath B) localization in membrane-disrupted and non-disrupted neurons within the lateral neocortex layers V and VI in sham-injured animals, animals sustaining a traumatic brain injury (TBI), and animals sustaining a TBI followed by secondary intracranial pressure (ICP) elevation (TBI + 20 mmHg ICP elevation) paired with 2w of intracerebroventricular infusion of 10% DMSO (vehicle) or the Cath B inhibitor CA-074Me. The left-most panel contains DAPI-labeled nuclei (blue). Neurons were identified as non-disrupted (yellow arrows) or membrane-disrupted (white arrowheads) based upon uptake of cell-impermeable Alexa-Fluor-488-tagged 10 kDa dextran (AF-dextran-488; second panel in green), which diffused throughout the parenchyma. Immunolabeling for Cath B (third panel in red) allowed investigation of Cath B localization inside lysosomal puncta or outside lysosomes. The right-most panel is an overlay of the single channel images. Scale bar 20 μm.

Article Snippet: The solid form of the Cath B inhibitor CA-074 methyl ester (CA-074Me; MedChemExpress, Cat#: HY-100350, Monmouth Junction, NJ, USA) was stored at −20 °C until reconstitution in 100% dimethylsulfoxide (DMSO) at a concentration of 100 µg/µL.

Techniques: Membrane, Labeling, Immunolabeling

Journal: Biomedicines

Article Title: The Effects of Cathepsin B Inhibition in the Face of Diffuse Traumatic Brain Injury and Secondary Intracranial Pressure Elevation

doi: 10.3390/biomedicines12071612

Figure Lengend Snippet: Cathepsin B (Cath B) inhibition with CA-074Me impacted the distribution of Cath B within lysosomal puncta. Bar graph depicting the percent of neurons within the non-disrupted (left half of graph; sham DMSO n = 214 neurons [dark green bars], sham CA-074Me n = 248 [light green bars], traumatic brain injury (TBI) DMSO n = 238 neurons [dark blue bars], TBI CA-074Me n = 275 neurons [light blue bars], TBI + intracranial pressure (ICP) DMSO n = 226 neurons [dark pink bars], and TBI + ICP CA-074Me n = 351 neurons [light pink bars]) and membrane-disrupted (right half of graph; sham DMSO n = 159 neurons [dark green bars], sham CA-074Me n = 176 neurons [light green bars], TBI DMSO n = 204 neurons [dark blue bars], TBI CA-074Me n = 205 neurons [light blue bars], TBI + ICP DMSO n = 153 neurons [dark pink bars], TBI + ICP CA-074Me n = 199 neurons [light pink bars]) populations that exhibited punctate localization of Cath B. Note that Cath B localization within puncta was impacted by inhibitor infusion, injury group, and membrane disruption status of the neurons analyzed. * p < 0.05 compared to non-disrupted counterpart, # p < 0.05 compared to sham and CA-074Me, $ p < 0.05 compared to vehicle-treated counterpart, ^ p < 0.05 compared to TBI-only infusion group counterpart. Mean ± S.E.M.

Article Snippet: The solid form of the Cath B inhibitor CA-074 methyl ester (CA-074Me; MedChemExpress, Cat#: HY-100350, Monmouth Junction, NJ, USA) was stored at −20 °C until reconstitution in 100% dimethylsulfoxide (DMSO) at a concentration of 100 µg/µL.

Techniques: Inhibition, Membrane, Disruption

Journal: Biomedicines

Article Title: The Effects of Cathepsin B Inhibition in the Face of Diffuse Traumatic Brain Injury and Secondary Intracranial Pressure Elevation

doi: 10.3390/biomedicines12071612

Figure Lengend Snippet: Somatosensory sensitivity was impacted by injury, particularly with secondary ICP elevation, and further impacted by cathepsin B inhibition with CA-074Me. Bar graph depicting the average whisker nuisance task (WNT) score pre-injury (tan bars) and at 2w post-injury or sham (blue bars). Sham animals infused with 10% DMSO (n = 11) had slightly higher WNT scores post-injury compared to sham animals infused with CA-074Me (n = 13). Animals sustaining traumatic brain injury (TBI) infused with 10% DMSO (n = 12) demonstrated similar post-injury WNT scores as TBI animals infused with CA-074Me (n = 13). Injured animals with secondary intracranial pressure (ICP) elevations (TBI + 20 mmHg ICP) infused with CA-074Me (n = 12) had lower WNT scores compared to injured and ICP-elevated animals infused with 10% DMSO (n = 11), resulting in an overall reduction in post-injury WNT score in animals infused with CA-074Me. * p < 0.05 compared pre-injury WNT score for that group, # p < 0.05 compared to sham 10% DMSO. Mean ± S.E.M.

Article Snippet: The solid form of the Cath B inhibitor CA-074 methyl ester (CA-074Me; MedChemExpress, Cat#: HY-100350, Monmouth Junction, NJ, USA) was stored at −20 °C until reconstitution in 100% dimethylsulfoxide (DMSO) at a concentration of 100 µg/µL.

Techniques: Inhibition, Whisker Assay

Journal: PLoS ONE

Article Title: Imaging Sites of Inhibition of Proteolysis in Pathomimetic Human Breast Cancer Cultures by Light-Activated Ruthenium Compound

doi: 10.1371/journal.pone.0142527

Figure Lengend Snippet: (A) Top view of representative 3D reconstruction of 16 contiguous fields of MDA-MB-231 breast carcinoma structures (nuclei, blue) and associated degradation fragments of DQ-collagen IV (green) at 4 days of culture. Panels from left to right are DMSO control and cysteine protease inhibitors (middle: 5 μM each of CA074 + CA074Me; right: uncaged inhibitor 1). (B) Hs578T breast carcinoma structures (nuclei, blue) and associated degradation fragments of DQ-collagen IV (green) at 4 days of culture. See A for further details. (C) Quantification of degraded DQ-collagen IV per cell in MDA-MB-231 (left) and Hs578T (right) structures exposed to DMSO (negative control), CA074/CA074Me (5 μM each; positive control) and uncaged inhibitor 1 . Data shown are from 3 independent experiments (48 fields); * ≤ 0.05; mean ± SD.

Article Snippet: Where indicated, assays were performed in the presence of CA074 and CA074Me (5 μM each) (Peptides International, Louisville, KY, USA), compound 1 or compound 2 .

Techniques: Control, Negative Control, Positive Control

Journal: PLoS ONE

Article Title: Imaging Sites of Inhibition of Proteolysis in Pathomimetic Human Breast Cancer Cultures by Light-Activated Ruthenium Compound

doi: 10.1371/journal.pone.0142527

Figure Lengend Snippet: Quantification of degraded collagen IV in entire 3D volume of MDA-MB-231 and Hs578T structures at 4 days of culture: total degraded collagen IV, black bars; pericellular degraded collagen IV, open bars; and intracellular degraded collagen IV, gray bars. DMSO (negative control), CA074/CA074Me (5 μM each; positive control) and uncaged inhibitor 1. Data shown are from 3 independent experiments (48 fields); * p < 0.05; mean ± SD.

Article Snippet: Where indicated, assays were performed in the presence of CA074 and CA074Me (5 μM each) (Peptides International, Louisville, KY, USA), compound 1 or compound 2 .

Techniques: Negative Control, Positive Control

Journal: Cancer Science

Article Title: Activation of ASC induces apoptosis or necrosis, depending on the cell type, and causes tumor eradication

doi: 10.1111/j.1349-7006.2010.01610.x

Figure Lengend Snippet: Necrotic cell death is prevented by cathepsin B inhibitor CA‐074Me. (a) The expression levels of cathepsin B (CathepB) and GAPDH in NUGC‐4, COLO205, NUC12N2, and CLC12N2 cell lines were examined by Western blotting. (b,c) The CLC12N2 cells were pretreated with the indicated inhibitors for 1 h, then stimulated with muramyl dipeptide (MDP; 100 ng/mL) for 8 h. The cell viability was assessed by WST‐1 assay. Experiments were done in duplicate, and error bars represent the range of the duplicate data. (−), no treatment; IETD, Z‐IETD‐fluoromethylketone.

Article Snippet: Soluble mouse Fas ligand was prepared as described previously. ( 18 ) Muramyl dipeptide (MDP; Sigma, St Louis, MO, USA), Z‐IETD‐fluoromethylketone (Z‐IETD‐FMK; R&D Systems, Minneapolis, MN, USA), CA‐074Me, cathepsin L inhibitor IV, cathepsin S inhibitor, calpain inhibitor XI (Merck Calbiochem, Tokyo, Japan), pepstatin A (Peptide Institute, Minoh, Osaka, Japan) and bafilomycin A (Fermentek, Jerusalem, Israel) were purchased.

Techniques: Expressing, Western Blot, WST-1 Assay

Journal: Cancer Science

Article Title: Activation of ASC induces apoptosis or necrosis, depending on the cell type, and causes tumor eradication

doi: 10.1111/j.1349-7006.2010.01610.x

Figure Lengend Snippet: Lysosomal leakage is involved in ASC‐mediated necrosis. (a) CLC12N2 cells stimulated with muramyl dipeptide (MDP; 100 ng/mL) for the indicated period were stained with acridine orange, and the red fluorescence was measured by flow cytometry (solid line). The dotted line indicates unstimulated cells. (b) CLC12N2 cells were pretreated with or without bafilomycin A1 (10 nM) or CA‐074Me (80 μM) for 1 h, then cultured in the presence or absence of MDP (100 ng/mL) for 3 h. The cells were stained with acridine orange and analyzed as described in (a). The dotted line indicates unstimulated cells. (c) CLC12N2 cells were pretreated with the indicated concentrations of bafilomycin A1 for 1 h, then stimulated without (−) or with MDP (100 ng/mL) for 8 h. The cell viability was assessed by WST‐1 assay. Experiments were done in duplicate, and error bars represent the range of the duplicate data.

Article Snippet: Soluble mouse Fas ligand was prepared as described previously. ( 18 ) Muramyl dipeptide (MDP; Sigma, St Louis, MO, USA), Z‐IETD‐fluoromethylketone (Z‐IETD‐FMK; R&D Systems, Minneapolis, MN, USA), CA‐074Me, cathepsin L inhibitor IV, cathepsin S inhibitor, calpain inhibitor XI (Merck Calbiochem, Tokyo, Japan), pepstatin A (Peptide Institute, Minoh, Osaka, Japan) and bafilomycin A (Fermentek, Jerusalem, Israel) were purchased.

Techniques: Staining, Fluorescence, Flow Cytometry, Cell Culture, WST-1 Assay