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

Journal: bioRxiv

Article Title: Hippocampal Glucuronyl C5-epimerase promotes stress resilience by directly engaging PI3K through a non-enzymatic mechanism

doi: 10.64898/2026.05.02.722405

Figure Lengend Snippet: ( A ) The docking model of the hPI3K-h Glce complex. ( B ) Direct binding between Glce and PI3Kα (p85α and p110α) proteins was measured by SPR. ( C ) Co-immunoprecipitation (Co-IP) showing the interaction between Glce and PI3Kα protein in primary cortical neurons and in hippocampal tissues by using anti- Glce , anti-PI3K p85α and p110α antibodies bidirectional pull-down. ( D ) PI3Kα co-localized with Glce in the Golgi apparatus in primary hippocampal neurons. Scale bars, 25 μm. ( E ) Relative PI3Kinase activity was examined in hippocampus tissues of NKO mice, n = 6 mice per group. ( F and G ) Western blot analysis of p-AKT/AKT protein level in the hippocampus tissues of NKO mice ( F ) and Glce or mut Glce overexpressed NKO mice ( G ) versus respective Glce fl/fl mice with quantification. ( H and ) Western blot (left) and quantification (right) showing the effect of Glce on CREB activation represented by p-CREB/CREB in the hippocampus of NKO mice ( H ) and Glce or mut Glce overexpressed NKO mice . ( J ) BDNF protein level was detected after treatment of KG501 (10 μM) and wortmannin (1 μM) in the primary cortical neurons (DIV3) from C57BL/6J mice for 24 hours by Western blot (left) with quantification (right). Error bars show s.e.m. Two-tailed unpaired t test ( E and F and H ) or one-way ANOVA, followed by Dunnett’s multiple comparisons test ( G and and J ) was used. *P < 0.05, **P < 0.01, ***P < 0.001. Note: , and were derived from the same Western blot experiment with common internal control GAPDH band.

Article Snippet: The PI3K enzymatic activity test was conducted according to the manufacturer’s instruction (GENMED, #GMS50058.2).

Techniques: Binding Assay, Immunoprecipitation, Co-Immunoprecipitation Assay, Activity Assay, Western Blot, Activation Assay, Two Tailed Test, Derivative Assay, Control

Journal: bioRxiv

Article Title: Hippocampal Glucuronyl C5-epimerase promotes stress resilience by directly engaging PI3K through a non-enzymatic mechanism

doi: 10.64898/2026.05.02.722405

Figure Lengend Snippet: ( A ) Schematic of stereotactic brain drug administration paradigm. ( B and C ) Bilateral infusion of LY294002 (10 μM) and 0.02% DMSO as solvent control via cannulae implantation in the HPC of 8-week-old male C57BL/6J mice, with dosing interval of 6 times in 3 weeks and then TST, SPT and LAT were employed to identify the effect of LY294002 on depressive-like behaviors. n = 12, 12 in the TST; n = 12, 12 in the SPT and n = 11, 12 in the LAT for Ctrl and LY294002 group, respectively. LY294002, a PI3K specific selective inhibitor ( B ). Golgi staining on brain slices from mice with LY294002 and 0.02% DMSO, with qualification of dendritic outgrowth (5-6 neurons per mouse). ( C ). Scale bars, 200 μm. ( D and E ) Bilateral infusion of 740 Y-P (30 μM) and saline as solvent control via cannulae implantation in the HPC of 8- to 12-week-old male NKO mice with dosing interval of 6 times in 2 weeks. Then TST; SPT and LAT were employed to identify the effect of 740 Y-P on NKO mice. n = 10, 12 in the TST; n = 11, 10 in the SPT and n = 12, 10 in the LAT for saline and 740 Y-P group, respectively. 740 Y-P, a PI3K specific selective agonist ( D ). Golgi staining on brain slices from NKO mice administrated with saline and 740 Y-P, with qualification of dendritic outgrowth. ( E ). Scale bars, 200 μm. Error bars show s.e.m. Two-tailed unpaired t test or Mann-Whitney test ( B to E ) were used. ns, not significant, *P < 0.05, **P < 0.01, ****P < 0.0001.

Article Snippet: The PI3K enzymatic activity test was conducted according to the manufacturer’s instruction (GENMED, #GMS50058.2).

Techniques: Solvent, Control, Staining, Saline, Two Tailed Test, MANN-WHITNEY

Journal: Plants

Article Title: Characterization of Endoglucanase (GH9) Gene Family in Tomato and Its Expression in Response to Rhizophagus irregularis and Sclerotinia sclerotiorum

doi: 10.3390/plants14223458

Figure Lengend Snippet: Schematic representation of the conserved protein motifs in tomato endoglucanase (SlGH9) genes. ( A ) Conserved motifs of SlGH9 proteins were identified. Distinctly colored boxes represented ten predicted motifs, and the gray lines indicated non-conserved regions. The sequence logo was created with 19 tomato endoglucanase SlGH9 protein sequences using the MEME program. ( B ) Sequence alignments of the catalytic conserved motif DGSEEGVDLVGGYYDAGDNVKFGFPMAFTTTMLSWSVIEYG in SlGH9 proteins.

Article Snippet: Endoglucanase enzymatic activity in AM colonized roots in lettuce [ ] and gene expression in Medicago truncatula [ ] and Lotus japonicus [ ] are induced, while their potential regulation in leaves remains unexplored.

Techniques: Sequencing

Journal: Plants

Article Title: Characterization of Endoglucanase (GH9) Gene Family in Tomato and Its Expression in Response to Rhizophagus irregularis and Sclerotinia sclerotiorum

doi: 10.3390/plants14223458

Figure Lengend Snippet: Expression patterns of endoglucanase SlGH9 genes in different tissues. Heat map representation of RPKM values (Log 2 ) for SlGH9 genes in tomato ( Solanum lycopersicum L.) vegetative tissues (seed, root, leaf, infected leaves with Sclerotinia Sclerotiorum (Lib.) DeBary and Botrytis cinerea (Pers.) at 0 and 30 h of infection) derived from RNA-seq data (SGN database) for S. lycopersicum cv. Heinz . White-colored cells indicate actual data values that are Log 2 -normalized to below 0. The color scale represents RPKM Log 2 values.

Article Snippet: Endoglucanase enzymatic activity in AM colonized roots in lettuce [ ] and gene expression in Medicago truncatula [ ] and Lotus japonicus [ ] are induced, while their potential regulation in leaves remains unexplored.

Techniques: Expressing, Infection, Derivative Assay, RNA Sequencing