Journal: International Journal of Biological Sciences

Article Title: Inhibition of Cathepsin B protects against vandetanib-induced hepato-cardiotoxicity by restoring lysosomal damage

doi: 10.7150/ijbs.122904

Figure Lengend Snippet: Vandetanib induced hepatocyte apoptosis by upregulating CTSB. ( A ) GO enrichment analysis of alterable protein expression after vandetanib's treatment. The red font highlighted parts mainly divided into apoptosis and lysosome pathways. ( B ) GSEA plots for apoptosis and lysosome KEGG pathways significantly enriched after vandetanib's treatment. ( C ) The mRNA levels of Ctsb and Ctsl in liver tissues were measured by qRT-PCR. (n = 5). ( D ) HL-7702 cells were treated with 0, 10, 20 and 30 μM vandetanib for 24 h. The mRNA levels of CTSB and CTSL were measured by qRT-PCR. (n = 3). ( E ) The protein levels of CTSB and CTSL in liver lysates were measured by western blot. (n = 6). ( F ) Human primary hepatocytes (HPH), murine primary hepatocytes (MPH), and AML12 cells were treated with 20 μM vandetanib for 0, 12, 24, 36 h or 0, 10, 20, 30 μM vandetanib for 36 h. The expression levels of c-PARP and CTSB were measured by western blot. ( G-I ) HL-7702 cells were transfected with vector, 0.5, 1 or 1.5 μg pcDNA3.0-CTSB-Flag plasmid for 36 h. ( G ) The survival rates were measured by SRB staining. (n = 3). ( H ) The apoptosis rates were detected by flow cytometry of Annexin V/PI staining. (n = 3). ( I ) The expression levels of c-PARP in total cell lysates were detected by western blot. ( J-K ) HL-7702 cells were transfected with negative control or siRNA targeting CTSB , and then treated with or without 20 μM vandetanib for 36 h. ( J ) The expression levels of c-PARP and CTSB in HL-7702 cells were measured by western blot. ( K ) The apoptosis rates were detected by flow cytometry of Annexin V/PI staining. Data are represented as the mean ± SD. ns, no significance, *p < 0.05, **p < 0.01, ***p < 0.001. Unpaired two-sided Student's t test for ( C ) and ( E ). One way ANOVA followed by Dunnett T3 post hoc test for ( D ), ( G ) and ( H ). One way ANOVA followed by Tukey post hoc test for ( K ).

Article Snippet: Cathepsin B (CTSB) enzymatic activity was measured using the fluorogenic substrate Z-Arg-Arg-AM Chydrochloride (HY-134434, MedChemExpress).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Transfection, Plasmid Preparation, Staining, Flow Cytometry, Negative Control

Journal: International Journal of Biological Sciences

Article Title: Inhibition of Cathepsin B protects against vandetanib-induced hepato-cardiotoxicity by restoring lysosomal damage

doi: 10.7150/ijbs.122904

Figure Lengend Snippet: Vandetanib induced lysosomal damage via CTSB-mediated cleavage of MCOLN1. ( A ) The expression levels of CTSB and c-MCOLN1 in liver tissues of mice. (n = 6). ( B ) The expression levels of p-AMPK Thr172/183 in liver tissues of mice. (n = 6). ( C ) The expression levels of Galectin-3 and LAMP1 in HL-7702 cells treated with 20 μM vandetanib for 36 h were measured by immunofluorescence. Scale bar, 20 μm. ( D-E ) HL-7702 cells were transfected with negative control or siRNA targeting CTSB , and then treated with 20 μM vandetanib for 36 h. ( D ) Representative images of Lyso-Tracker staining in HL-7702. Scale bar, 20 μm. ( E ) The expression levels of Galectin-3 and LAMP1 in HL-7702 cells were measured by immunofluorescence. Scale bar, 20 μm. ( F ) HL-7702 cells were treated with 20 μM vandetanib for 36 h. The expression levels of LC3-II in HL-7702 cells were measured by immunofluorescence. Scale bar, 25 μm. ( G ) The expression levels of SQSTM1 and LC3-I/II in HL-7702 cells treated with 20 μM vandetanib for 0, 10, 20, 30 μM vandetanib for 36 h were detected by western blot. ( H ) The expression levels of LC3-II and SQSTM1 in liver tissues of mice were detected by immunohistochemical analysis. Scale bar, 100 μm. ( I ) Representative confocal fluorescence micrographs of HL-7702 cells transfected with Ad-mCherry-GFP-LC3B and treated with 0, 10, 20, 30 μM vandetanib for 24 h. Scale bar, 20 μm. ( J ) The expression levels of c-PARP and LC3-I/II in HL-7702 cells treated with 20 μM vandetanib for 0, 3, 6, 9, 12 and 24 h were detected by western blot. Data are represented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. Unpaired two-sided Student's t test for ( A ), ( B ) and ( H ).

Article Snippet: Cathepsin B (CTSB) enzymatic activity was measured using the fluorogenic substrate Z-Arg-Arg-AM Chydrochloride (HY-134434, MedChemExpress).

Techniques: Expressing, Immunofluorescence, Transfection, Negative Control, Staining, Western Blot, Immunohistochemical staining, Fluorescence

Journal: International Journal of Biological Sciences

Article Title: Inhibition of Cathepsin B protects against vandetanib-induced hepato-cardiotoxicity by restoring lysosomal damage

doi: 10.7150/ijbs.122904

Figure Lengend Snippet: Knockdown of CTSB ameliorated vandetanib-induced hepatotoxicity in vivo. ( A-D ) C57BL/6 mice were randomly divided into 4 groups. After injection of AAV9-sh Ctsb adeno virus by tail vein for 3 weeks, mice were treated with 0.5% CMC-Na or 100 mg/kg/day vandetanib by gavage for another 4 weeks. ( A ) (Left panel) Representative photographs of mice liver. (Right panel) Representative images of H&E staining in liver tissues. Scale bar, 100 μm. ( B ) The levels of serum ALT and AST. (n = 8). ( C ) The expression levels of cleaved Caspase 3, p-AMPK Thr172/183, CTSB and LC3-II in liver tissues were detected by immunohistochemical analysis. Scale bar, 100 μm. ( D ) The expression levels of c-PARP, p-AMPK Thr172/183, AMPK, CTSB and LC3-I/II in liver tissues were measured by western blot. (n = 4). ( E ) Representative images of TUNEL staining in liver tissues. Scale bar, 100 μm. Data are represented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. One way ANOVA followed by Tukey post hoc test for ( B ), ( C ), ( D ) and ( E ).

Article Snippet: Cathepsin B (CTSB) enzymatic activity was measured using the fluorogenic substrate Z-Arg-Arg-AM Chydrochloride (HY-134434, MedChemExpress).

Techniques: Knockdown, In Vivo, Injection, Virus, Staining, Expressing, Immunohistochemical staining, Western Blot, TUNEL Assay

Journal: International Journal of Biological Sciences

Article Title: Inhibition of Cathepsin B protects against vandetanib-induced hepato-cardiotoxicity by restoring lysosomal damage

doi: 10.7150/ijbs.122904

Figure Lengend Snippet: Tannic acid inhibited vandetanib-induced hepatocyte death by direct binding to CTSB. ( A-B ) HL-7702 cells were treated with 20 μM vandetanib and/or 5 μM CA-074, 5 μM Apigenin, 5 μM Baicalein, 5 μM Tannic acid for 36 h. ( A ) The survival rates of HL-7702 cells were measured by SRB staining. (n = 6). ( B ) The expression levels of c-PARP and p-AMPK Thr172/183 were analyzed by western blot. ( C ) Molecular docking of tannic acid and CTSB. ( D ) The binding stability determined by CETSA assay of drug molecules to proteins. ( E ) HL-7702 cells were treated with 20 μM vandetanib and/or 5 μM tannic acid for 36 h. Representative images of Lyso-Tracker staining in HL-7702 cells. Scale bar, 20 μm. ( F ) The expression levels of Galectin-3 and LAMP1 in HL-7702 cells treated with 20 μM vandetanib for 36 h were measured by immunofluorescence. Scale bar, 20 μm. ( G ) HL-7702 cells were treated with 20 μM vandetanib and/or 5 μM tannic acid for 36 h. The expression levels of c-PARP and c-MCOLN1 were measured by western blot. ( H ) HL-7702 cells were treated with 20 μM vandetanib and/or 5 μM tannic acid for 36 h. The apoptosis rates were detected by flow cytometry of Annexin V/PI staining. (n = 3). ( I ) Autophagic flux was assessed in HL-7702 cells transfected with Ad-mCherry-GFP-LC3B using confocal microscopy. (Upper) Cells were treated with 20 μM vandetanib and/or 5 μM tannic acid for 36 h. (Lower) Cells were transfected with negative control or CTSB-targeting siRNA followed by treatment with or without 20 μM vandetanib for 36 h. Scale bar, 10 μm. Data are represented as the mean ± SD. ***p < 0.001. One way ANOVA followed by Tukey post hoc test for ( A ) and ( H ).

Article Snippet: Cathepsin B (CTSB) enzymatic activity was measured using the fluorogenic substrate Z-Arg-Arg-AM Chydrochloride (HY-134434, MedChemExpress).

Techniques: Binding Assay, Staining, Expressing, Western Blot, Immunofluorescence, Flow Cytometry, Transfection, Confocal Microscopy, Negative Control

Journal: International Journal of Biological Sciences

Article Title: Inhibition of Cathepsin B protects against vandetanib-induced hepato-cardiotoxicity by restoring lysosomal damage

doi: 10.7150/ijbs.122904

Figure Lengend Snippet: Targeting CTSB alleviated vandetanib-induced cardiac injury. ( A-E ) C57BL/6 mice were randomly divided into 4 groups (n = 8 per group). After injection of AAV9-sh Ctsb adeno virus by tail vein for 3 weeks, mice were treated with 0.5% CMC-Na or 100 mg/kg/day vandetanib by gavage for another 4 weeks. ( A ) Representative M-mode echocardiogram images. ( B ) Quantifications of Ejection fraction and Fractional shortening ratios. ( C ) (Upper photos) Representative photographs of heart tissues. Scale bar, 500 μm. (Lower photos) H&E staining of heart tissues. Scale bar, 50 μm. ( D ) Representative images of immunohistochemistry for cleaved Caspase 3 staining in heart tissues. Scale bar: 50 μm. ( E ) Total RNA was extracted from mice hearts and the expression levels of Myh6 , Myh7 , Nppa and Nppb were measured by qRT-PCR. ( F-J ) C57BL/6 mice were randomly divided into 4 groups (n = 6 per group). The C57BL/6J mice were received 100 mg/kg vandetanib and/or 30 mg/kg tannic acid for 4 weeks. ( F ) Representative M-mode echocardiogram images. ( G ) Quantifications of Ejection fraction and Fractional shortening ratios. ( H ) (Upper photos) Representative photographs of heart tissues. Scale bar, 500 μm. (Lower photos) H&E staining of heart tissues. Scale bar, 50 μm. ( I ) Representative images of immunohistochemistry for cleaved Caspase 3 staining in heart tissues. Scale bar: 50 μm. ( J ) Total RNA was extracted from mice hearts and the expression levels of Myh6 , Myh7 , Nppa and Nppb were measured by qRT-PCR. Data are represented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. One way ANOVA followed by Tukey post hoc test for ( B ), ( E ), ( G ) and ( J ).

Article Snippet: Cathepsin B (CTSB) enzymatic activity was measured using the fluorogenic substrate Z-Arg-Arg-AM Chydrochloride (HY-134434, MedChemExpress).

Techniques: Injection, Virus, Staining, Immunohistochemistry, Expressing, Quantitative RT-PCR