Journal: mBio

Article Title: Cathepsin B hyperactivation facilitates exosome release of CVB3 particles and exacerbation of acute pancreatitis by impairing lysosomal integrity and acidification

doi: 10.1128/mbio.03111-25

Figure Lengend Snippet: CTSB increases viral replication and virion release in pancreatic acinar cells. ( A ) Immunoblotting analysis of CTSB and VP1 proteins in 266-6 cells and SW1990 cells infected with CVB3 (MOI = 1) for 1 h, and treated with CTSB inhibitor, CA074Me for 12 h. Densitometric analysis of CTSB normalized to tubulin is shown as fold change. ( B ) Confocal microscopy of 266-6 cells infected with eGFP-CVB at MOI = 1 for 1 h and treated with 20 µM CA074Me for 12 h. Scale bars represent 100 µm. ( C ) Quantification of CVB3 titer in CA074Me-treated 266-6 cell culture supernatant by plaque-forming assay or mRNA levels by real-time qPCR. ( D ) Immunoblotting analysis of CTSB and VP1 proteins in 266-6 and SW1990 cells pre-treated with siRNA-CTSB for 36 h, then infected with CVB3 (MOI = 1) for 12 h. ( E ) Plaque-forming assay of CVB3 titer in cell culture or real-time qPCR analysis of CVB3 mRNA levels in 266-6 cells. ( F ) Immunoblotting analysis of CTSB and VP1 proteins in 266-6 cells pre-transfected with pCDH or pCTSB plasmid for 36 h, then infected with CVB3 (MOI = 1) for 1 h, maintained for 12 h. ( G ) CVB3 titer in culture supernatant and viral mRNA expression were determined by plaque forming assay and real-time qPCR. Data from panels C, E, and G were shown as mean ± SEM of averages from three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Cells with 85% confluency were infected with CVB3 at a multiplicity of infection (MOI) of 1, 5, or 10 for 1 h. After washing with PBS, cells were maintained for 12 ~ 24 h. In experiments where CA074Me (Cathepsin B Inhibitor) (TargetMol T3420) was used, cells were treated with 10 μM CA074-Me dissolved in DMSO (Sigma) for 12 h after CVB3 infection (MOI of 1).

Techniques: Western Blot, Infection, Confocal Microscopy, Cell Culture, Transfection, Plasmid Preparation, Expressing

Journal: mBio

Article Title: Cathepsin B hyperactivation facilitates exosome release of CVB3 particles and exacerbation of acute pancreatitis by impairing lysosomal integrity and acidification

doi: 10.1128/mbio.03111-25

Figure Lengend Snippet: Inhibition of CTSB blocks exosome release of CVB3 via rescuing lysosome integrity and function after infection. 266-6 cells were infected with CVB3 (MOI = 1) for 1 h, washed, and then treated with CA074Me (20 µM) for 12 h. ( A ) Immunoblotting analysis of CTSB and CTSD in cell lysosomal and cytoplasmic fractions lysates. Densitometric analysis of CTSB/CTSD normalized to GAPDH or LAMP1 is shown as fold changes compared with control. ( B ) CTSB activity of CA074Me-treated 266-6 cells. ( C ) Fluorescent microscopy images of control and CA074Me-treated cells after CVB3 infection (MOI = 1, for 12 h) labeled with Lysotracker Red (LTR). Mean fluorescent intensity (MFI) of cells was quantified from n = 25 cells per section. Arrow indicates Lysotracker Red positive staining. Scale bars: 10 µm. ( D ) mRNA expressions of ctsb , Mcoln1 , Atp6v1h , and M6pr in CA074Me-treated cells were analyzed by real-time PCR. ( E ) CCK-8 assay of cell viability 12 h after CA074Me treatment. Data were expressed as mean ± SEM of averages from three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Cells with 85% confluency were infected with CVB3 at a multiplicity of infection (MOI) of 1, 5, or 10 for 1 h. After washing with PBS, cells were maintained for 12 ~ 24 h. In experiments where CA074Me (Cathepsin B Inhibitor) (TargetMol T3420) was used, cells were treated with 10 μM CA074-Me dissolved in DMSO (Sigma) for 12 h after CVB3 infection (MOI of 1).

Techniques: Inhibition, Infection, Western Blot, Control, Activity Assay, Microscopy, Labeling, Staining, Real-time Polymerase Chain Reaction, CCK-8 Assay

Journal: mBio

Article Title: Cathepsin B hyperactivation facilitates exosome release of CVB3 particles and exacerbation of acute pancreatitis by impairing lysosomal integrity and acidification

doi: 10.1128/mbio.03111-25

Figure Lengend Snippet: Inhibition of CTSB blocks exosomal release of virus. 266-6 cells were infected with CVB3 (MOI = 1) for 1 h, maintained in the presence of CA074me (20 µM) for 12 h. ( A ) BCA assay analysis of exosomes harvested from CVB3-infected 266-6 cells at indicated times. ( B ) Immunoblotting analysis of exosomal markers (CD9, CD63, and Alix) expression in exosomes purified from supernatant of 266-6 cells infected with CVB3 for 1 h and treated with bafilomycin A1 (20 nM) for 12 h. ( C ) Relative fluorescence units (RFU) detection of GFP + virus in exosomes from 266 to 6 cells infected with GFP-CVB3 (MOI = 1). ( D ) BCA assay of exosomal protein levels harvested from culture supernatant of infected 266-6 cells. ( E ) Western blot detection of exosomal markers CD9, CD63, and Alix expression in exosome lysates. ( F ) Relative fluorescence units (RFU) detection of eGFP-CVB3 progeny in 266-6 cells after CVB3 infection (MOI = 1) for 24 h. ( G ) Viral titers of 266-6 or SW1990 cells infected with 0.1 µg purified exosomes (from infected 266-6 cells treated with CA074Me). ( H, J ) Plaque assay determination of viral titers of exosome lysate from 1 mL supernatant of 266-6 cells treated with CA074Me ( H ), or from cells transfected with con-siRNA or CTSB-siRNA 36 h before CVB3 (MOI = 1) infection ( J ). Total viral titer: viral titer in whole cell culture supernatant; exosome viral titer: free viral titer in the exosome-removed supernatant. ( I ) Western blot analysis of CTSB and VP1 levels in 266-6-exosome and whole cell lysate (WCL). Data from panels A, C, D, F, G, H, and J were shown as mean ± SEM of averages from three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Cells with 85% confluency were infected with CVB3 at a multiplicity of infection (MOI) of 1, 5, or 10 for 1 h. After washing with PBS, cells were maintained for 12 ~ 24 h. In experiments where CA074Me (Cathepsin B Inhibitor) (TargetMol T3420) was used, cells were treated with 10 μM CA074-Me dissolved in DMSO (Sigma) for 12 h after CVB3 infection (MOI of 1).

Techniques: Inhibition, Virus, Infection, BIA-KA, Western Blot, Expressing, Purification, Fluorescence, Plaque Assay, Transfection, Cell Culture

Journal: mBio

Article Title: Cathepsin B hyperactivation facilitates exosome release of CVB3 particles and exacerbation of acute pancreatitis by impairing lysosomal integrity and acidification

doi: 10.1128/mbio.03111-25

Figure Lengend Snippet: Therapeutic administration of CTSB inhibitor reduces viral infection and AP pathology in mice. ( A ) Schematic map of CA074Me treatment experiment. Mice were i.p. injected with 10.5 mg/Kg CA074Me on day 1 and day 2 after 10 3 pfu CVB3 infection on day 0. ( B ) Survival curve and weight loss curve of sham, CVB3, and CA074Me-treated mice were followed by 7 dpi. ( C ) Representative images of H&E staining of pancreatic tissues. Arrows indicate lymphocyte infiltration. Scale bar: 100 µm. ( D ) mRNA levels of Il-6 , Il-1β , Il-17a , and Tnf-α from day 7 mice pancreas were analyzed by real-time qPCR. ( E ) Immunoblotting analysis of day 3 pancreas total lysates for VP1 expression. ( F ) Plaque assay on day 3 pancreas homogenates to determine viral titer. Viral mRNA levels were analyzed by real-time qPCR. Data from panels C, D, E, and F were shown as mean ± SEM of averages from three independent experiments. * P < 0.05, **< 0.01, *** P < 0.001.

Article Snippet: Cells with 85% confluency were infected with CVB3 at a multiplicity of infection (MOI) of 1, 5, or 10 for 1 h. After washing with PBS, cells were maintained for 12 ~ 24 h. In experiments where CA074Me (Cathepsin B Inhibitor) (TargetMol T3420) was used, cells were treated with 10 μM CA074-Me dissolved in DMSO (Sigma) for 12 h after CVB3 infection (MOI of 1).

Techniques: Infection, Injection, Staining, Western Blot, Expressing, Plaque Assay

Journal: The Journal of Physiology

Article Title: Intracellular rupture, exocytosis and actin interaction of endocytic vacuoles in pancreatic acinar cells: initiating events in acute pancreatitis

doi: 10.1113/JP275879

Figure Lengend Snippet: A , cells were stimulated by 10 n m CCK for 2 h at 35 °C in the presence of diS‐Cy5 and dextran Texas Red 3000 MW neutral (TRD). Cells were then washed by perfusion with standard extracellular solution. Following the wash, cells were immersed in the extracellular solution containing FITCD and imaged. Upper left: transmitted light image of the cells. Scale bar = 10 μm. Images of the fluorescence of the cells and extracellular milieu recorded using excitation/emission corresponding to the specified probes shown elsewhere. Note the EV in the right cell and increased cytosolic fluorescence on diS‐Cy5 and TRD images of the left cell. The FITCD image indicates that the plasma membrane of the cell is intact. B , representative fluorescence emission spectra recorded from cells stimulated as in ( A ) but in the presence of diS‐Cy5 only. The fluorescence was excited by a 595 nm laser line. The graphs show fluorescence spectra recorded from an EV (green), cytosol of a cell that satisfies criteria for detecting the EV's rupture/leakage (red; see Methods and C ), cytosol of a cell that did not satisfy criteria for detecting rupture/leakage (black; see Methods and C ). The blue trace in the insert is produced by subtracting black trace (mainly determined by cellular autofluorescence) from the cytosolic fluorescence of a cell that satisfies the criteria for detecting rupture/leakage. The residual trace shows a spectrum that is similar to the spectrum of diS‐Cy5 recorded from an EV. The red and black traces were recorded from different cells and shown on the same graph for illustration. C , left: intensities of cytosolic fluorescence in cells that were immersed in the indicator‐containing diS‐Cy5 solution for 30 min but were not stimulated. Only a very few small vacuoles are expected to form during this period of time (Fig. B and C ) and the distribution should therefore reflect cytosolic fluorescence in the cells that did not have ruptured EVs. The blue trace represents a single Gaussian approximation of the distribution. Right: frequency histogram of cells after two hours of incubation with diS‐Cy5. The CCK concentration was 10 n m . The first two Gaussian peaks of the approximation are shown by blue and magenta lines. Cells with cytosolic fluorescence above threshold (central value of the first peak plus 3 sigma) are classified as the cells that experienced rupture/leakage of EV(s). D , the method illustrated in ( A ) to ( C ) was used to evaluate the proportions of cells with ruptured vacuoles. CCK concentration was 10 n m (in specified experiments). Neither inhibition of serine protease with benzamidine (1 m m ), nor inhibition of cathepsin B with combination of CA074 (10 μ m ) and CA074‐Me (1 μ m ) (abbreviated as CA074/Me) produced a significant difference in the proportion of cells with increased cytosolic fluorescence from control. Inhibition of V‐ATPase with 100 n m of bafilomycin A1 (Baf) also did not produce a statistically significant change in the proportion of cells with increased cytosolic fluorescence. The number of experiments in each condition was: n = 20 experiments for control (unstimulated cells) and CCK; n = 9 for CA074/Me and CA074/Me + CCK; n = 8 for benzamidine and benzamidine + CCK; n = 6 for bafilomycin A1 and bafilomycin A1 + CCK. Each of the individual experiments involved acquisition and analysis of a fluorescence distribution similar to that shown on the right of ( C ).

Article Snippet: CA‐074 and bafilomycin A1 were obtained from Tocris Bioscience (Bristol, UK) and CA‐074Me was obtained from Calbiochem.

Techniques: Fluorescence, Clinical Proteomics, Membrane, Produced, Incubation, Concentration Assay, Inhibition, Control

Journal: The Journal of biological chemistry

Article Title: Negative regulation of epidermal growth factor signaling by selective proteolytic mechanisms in the endosome mediated by cathepsin B.

doi: 10.1074/jbc.274.47.33723

Figure Lengend Snippet: FIG. 4. Effect of pH and protease inhibitors on the endosomal EGF-degrading activity. Soluble endosomal extract (about 1 ng) was incubated with 1026 M EGF at 37 °C for 1 h in 50 mM citrate-phosphate buffer at the indicated pH (A) and for 1 h in 50 mM citrate-phosphate buffer, pH 5, in the absence or presence of 1 mM EDTA, 1 mM 1,10- phenanthroline, 1 mM phenylmethylsulfonyl fluoride, 10 mg/ml leupep- tin, 1027 M E64, 1027 M CA074-Me, or 10 mg/ml pepstatin A (B). At the end of the incubation, the degradation of EGF was measured by HPLC analysis as described in Fig. 2. Results for B are expressed as EGF degraded (percentage of control) after a 1-h incubation and normalized (100%) to that seen in the absence of added compound. Results are the mean 6 SD of three different experiments performed on endosomal fractions prepared from separate liver fractionations.

Article Snippet: CA074-Me, the methyl ester of CA074, which is a highly selective irreversible cathepsin B inhibitor, was purchased from Peptides International.

Techniques: Activity Assay, Incubation, Control

Journal: The Journal of biological chemistry

Article Title: Negative regulation of epidermal growth factor signaling by selective proteolytic mechanisms in the endosome mediated by cathepsin B.

doi: 10.1074/jbc.274.47.33723

Figure Lengend Snippet: FIG. 9. Effect of CA074-Me treatment on the uptake of [125I]EGF in rat liver. Rats were injected with [125I]EGF (107 cpm) having first received an intraperitoneal injection of either 0.15 M NaCl (open bars) or 0.5 mmol CA074-Me (closed bars) 1 h prior to ligand administration. Liver homogenates (A) and endosomal fractions (B) were prepared from rats sacrificed at the indicated times. Recovered radioactivity in the homogenates and endosomal fractions was quanti- fied, and the enrichment of internalized [125I]EGF is expressed by reference to the radioactivity in the homogenates (relative specific activity). The results are means 6 S.D. from three to five fractionations.

Article Snippet: CA074-Me, the methyl ester of CA074, which is a highly selective irreversible cathepsin B inhibitor, was purchased from Peptides International.

Techniques: Injection, Radioactivity, Activity Assay

Journal: The Journal of biological chemistry

Article Title: Negative regulation of epidermal growth factor signaling by selective proteolytic mechanisms in the endosome mediated by cathepsin B.

doi: 10.1074/jbc.274.47.33723

Figure Lengend Snippet: FIG. 10. Effect of CA074-Me treat- ment on the internalization of tyro- sine-phosphorylated EGFR following administration of EGF in vivo. Rats were injected with native EGF (5 mg/100 g of body weight), having first received an intraperitoneal injection of either 0.15 M NaCl or 0.5 mmol of CA074-Me 1 h prior to ligand administration. Endosomal frac- tions were isolated at the indicated times and evaluated by Western blotting for their content of EGFR and SHC by using their respective polyclonal IgG and for their immunoreactivity with monoclonal antibody against phosphotyrosine. Each lane contained 20 mg of protein of the endosomal fraction. The arrows on the right indicate the mobility of the EGFR (about 170 kDa) and the three isoforms of SHC (about 66, 55, and 46 kDa). The po- sitions of molecular mass markers are shown at the left.

Article Snippet: CA074-Me, the methyl ester of CA074, which is a highly selective irreversible cathepsin B inhibitor, was purchased from Peptides International.

Techniques: In Vivo, Injection, Isolation, Western Blot

Journal: The Journal of biological chemistry

Article Title: Negative regulation of epidermal growth factor signaling by selective proteolytic mechanisms in the endosome mediated by cathepsin B.

doi: 10.1074/jbc.274.47.33723

Figure Lengend Snippet: FIG. 12. Effect of protease inhibitors on the endosomal EGFR- degrading activity. Rat hepatic endosomal fractions were isolated 15 min after EGF administration (5 mg/100 g of body weight) and incu- bated in isotonic buffer at 37 °C and pH 5 in the absence or presence of 1027 M E64, 1027 M CA074-Me, 10 mg/ml leupeptin, 1 mM EDTA, 0.1 mM (p-hydroxymercuribenzoate), 0.1 mM (p-hydroxymercuriphenylsulfonic acid), 1 mM iodoacetic acid, or 10 mg/ml pepstatin A. After a 2 min incubation, the integrity of the endosome-associated EGFR was evalu- ated by Western blotting. Each lane contains 20 mg of protein of the endosomal fraction. The positions of molecular mass markers are shown at the left.

Article Snippet: CA074-Me, the methyl ester of CA074, which is a highly selective irreversible cathepsin B inhibitor, was purchased from Peptides International.

Techniques: Activity Assay, Isolation, Incubation, Western Blot

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