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