enzymatic shearing kit  (Active Motif)

Journal: Journal of Advanced Research

Article Title: Andrographolide alleviates type 2 diabetic nephropathy through suppressing PI3K/AKT1/RRM2-triggered oxeiptosis

doi: 10.1016/j.jare.2025.10.070

Figure Lengend Snippet: Upregulation of PI3K/AKT1 Pathway, RRM2, and oxeiptosis in type 2 diabetic nephropathy (T2DN). (A) Renal transmission electron micrographs of each groups in animal studies. Scale bar: 2 μm and zoomed scale bar: 500 nm. (B) Schematic illustration of the core scientific questions in this study. (C) Expression levels of CAT and GPX1 in animal experimental groups. (D) Quantitative analysis of CAT and GPX1 protein levels based on (C). (E) Hydrogen peroxide levels in renal tissues. (F) Reactive oxygen species (ROS) levels in renal tissues. (G) Hydrogen peroxide levels in MDCK cells treated with 50 mM glucose (GLU) and the regulatory effects of andrographolide (AND). (H) ROS levels in MDCK cells treated with 50 mM GLU and the regulatory effects of AND. Scale bar: 100 μm. (I) Quantitative analysis of ROS levels based on (H). (J) Protein expression levels of PI3K/AKT1 pathway, RRM2, and oxeiptosis-related proteins in renal tissues. (K) Quantitative analysis of target protein expression levels based on (J). (L) Immunohistochemical (IHC) staining of p-AKT1 (PI3K/AKT1 pathway), RRM2, and oxeiptosis-related proteins. Scale bar: 200 μm. (M) Quantitative analysis of protein expression levels based on (L). (N) Expression profiles of oxeiptosis-related proteins in MDCK cells exposed to 50 mM GLU. (O) Quantitative analysis of oxeiptosis-related protein levels based on (N). (P) Effects of EUK-134 on oxeiptosis in MDCK cells treated with 50 mM GLU. (Q) Quantitative analysis of oxeiptosis-related protein levels based on (P). All data are presented as mean ± SD, n = 3.

Article Snippet: Moreover, Z-VAD-FMK (Z-VAD; HY-16658B, MCE, China) was used to inhibit pan-caspase activity, Necrostatin-1 (Nec-1; HY-15760, MCE, China) to inhibit necroptosis, and Hydroxyurea (HU; HY-B0313, MCE, China) to inhibit RRM2 enzymatic activity.

Techniques: Transmission Assay, Expressing, Immunohistochemical staining, Immunohistochemistry

Journal: Journal of Advanced Research

Article Title: Andrographolide alleviates type 2 diabetic nephropathy through suppressing PI3K/AKT1/RRM2-triggered oxeiptosis

doi: 10.1016/j.jare.2025.10.070

Figure Lengend Snippet: PI3K/AKT1 mediates H 2 O 2 -induced oxeiptosis, while andrographolide (AND) reverses hyperglycemia-triggered upregulation of PI3K/AKT1 signaling, RRM2, and oxeiptosis. (A) Effect of miltefosine on hydrogen peroxide-induced alterations in the PI3K/AKT1 signaling pathway, RRM2 expression, and oxeiptosis-associated proteins. (B) Quantitative analysis of protein levels shown in (A). (C) Phosphorylation kinetics of the PI3K/AKT pathway in response to H 2 O 2 . (D) Molecular docking between H 2 O 2 and PI3K. (E) Expression profiles of PI3K/AKT1 pathway components, RRM2, and oxeiptosis markers under 50 mM glucose (GLU) stimulation. (F) Quantitative analysis of protein levels shown in (E). (G) Pan-caspase inhibitor Z-VAD-FMK (Z-VAD) and necroptosis inhibitor Necrostatin-1 (Nec-1) fail to completely reverse apoptosis induced by H 2 O 2 -triggered oxeiptosis. (H) Apoptosis detection using Annexin V-mCherry/SYTOX Green staining in cells exposed to 50 mM GLU. (I) Quantification of Annexin V-mCherry-positive expression from (H). (J) GLU-induced mitochondrial co-localization and expression of AIFM1 (pS116). (K) Quantitative analysis of AIFM1 (pS116) expression from (J). (L) AND-mediated restoration of PI3K/AKT1 signaling, RRM2 levels, and oxeiptosis regulation under GLU stimulation. (M) Quantitative analysis of protein levels shown in (L). (N) AND-dependent attenuation of GLU-induced apoptosis detected by Annexin V-mCherry/SYTOX Green. (O) Quantification of Annexin V-mCherry-positive expression from (N). (P) AND-mediated modulation of AIFM1 (pS116) expression and its co-localization with mitochondrial under GLU stress. (Q) Quantitative analysis of AIFM1 (pS116) expression from (P). (R) AND suppresses H 2 O 2 upregulation and modulates the high GLU-promoted PI3K/AKT1 pathway, RRM2 expression, and oxeiptosis. Scale bar: 10 µM. All data are presented as mean ± SD, n = 3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Moreover, Z-VAD-FMK (Z-VAD; HY-16658B, MCE, China) was used to inhibit pan-caspase activity, Necrostatin-1 (Nec-1; HY-15760, MCE, China) to inhibit necroptosis, and Hydroxyurea (HU; HY-B0313, MCE, China) to inhibit RRM2 enzymatic activity.

Techniques: Expressing, Phospho-proteomics, Staining

Journal: Journal of Advanced Research

Article Title: Andrographolide alleviates type 2 diabetic nephropathy through suppressing PI3K/AKT1/RRM2-triggered oxeiptosis

doi: 10.1016/j.jare.2025.10.070

Figure Lengend Snippet: Regulation of oxeiptosis by the PI3K/AKT1 pathway and RRM2 (A) Effect of AKT1 overexpression on 50 mM glucose (GLU)-modulated PI3K/AKT1 pathway activity, RRM2 expression, and oxeiptosis-associated proteins. (B) Quantitative analysis of protein levels in (A). (C) Apoptosis detection via Annexin V-mCherry/SYTOX Green staining under AKT1 overexpression and GLU treatment. (D) Quantification of Annexin V-mCherry-positive expression from (C). (E) AKT1 overexpression modulates AIFM1 (pS116) phosphorylation and mitochondrial co-localization during GLU exposure. (F) Quantitative analysis of AIFM1 (pS116) levels in (E). (G) Impact of AKT1 knockdown on GLU-driven PI3K/AKT1 signaling, RRM2 expression, and oxeiptosis markers. (H) Quantitative analysis of protein levels in (G). (I) AKT1 knockdown attenuates GLU-induced apoptosis detected by Annexin V-mCherry/SYTOX Green. (J) Quantification of Annexin V-mCherry-positive expression from (I). (K) AKT1 inhibition-mediated modulation of AIFM1 (pS116) expression and its co-localization with mitochondrial under GLU stress. (L) Quantitative analysis of AIFM1 (pS116) levels in (K). (M) Effect of RRM2 overexpression on 50 mM GLU-modulated PI3K/AKT1 pathway activity, RRM2 expression, and oxeiptosis-associated proteins. (N) Quantitative analysis of protein levels in (M). (O) Apoptosis detection via Annexin V-mCherry/SYTOX Green staining under RRM2 overexpression and GLU treatment. (P) Quantification of Annexin V-mCherry-positive expression from (O). (Q) RRM2 overexpression modulates AIFM1 (pS116) phosphorylation and mitochondrial co-localization during GLU exposure. (R) Quantitative analysis of AIFM1 (pS116) levels in (Q). (S) Impact of RRM2 inhibition on GLU-driven PI3K/AKT1 signaling, RRM2 expression, and oxeiptosis markers. (T) Quantitative analysis of protein levels in (S). (U) RRM2 knockdown attenuates GLU-induced apoptosis detected by Annexin V-mCherry/SYTOX Green. (V) Quantification of Annexin V-mCherry-positive expression from (U). (W) RRM2 inhibition-mediated modulation of AIFM1 (pS116) expression and its co-localization with mitochondrial under GLU stress. (X) Quantitative analysis of AIFM1 (pS116) levels in (W). Scale bar: 10 µM. All data are presented as mean ± SD, n = 3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Moreover, Z-VAD-FMK (Z-VAD; HY-16658B, MCE, China) was used to inhibit pan-caspase activity, Necrostatin-1 (Nec-1; HY-15760, MCE, China) to inhibit necroptosis, and Hydroxyurea (HU; HY-B0313, MCE, China) to inhibit RRM2 enzymatic activity.

Techniques: Over Expression, Activity Assay, Expressing, Staining, Phospho-proteomics, Knockdown, Inhibition

Journal: Journal of Advanced Research

Article Title: Andrographolide alleviates type 2 diabetic nephropathy through suppressing PI3K/AKT1/RRM2-triggered oxeiptosis

doi: 10.1016/j.jare.2025.10.070

Figure Lengend Snippet: High glucose (GLU) activates oxeiptosis through the PI3K/AKT1/RRM2 axis. (A) Interaction between RRM2 and AKT1 in HEK293T cells. (B) Protein-protein docking of RRM2 and AKT1. (C) Effects of RRM2 overexpression combined with Recilisib on the PI3K/AKT1 pathway, RRM2 expression, and oxeiptosis-associated proteins under 50 mM GLU treatment. (D) Quantitative analysis of PI3K/AKT1 pathway activity, RRM2 expression, and oxeiptosis-related protein levels in (C). (E) Apoptosis detection via Annexin V-mCherry/SYTOX Green staining in RRM2-overexpressing cells treated with Recilisib and 50 mM GLU. (F) Quantitative analysis of Annexin V-mCherry-positive expression from (E). (G) RRM2 overexpression combined with Recilisib modulates AIFM1 (pS116) phosphorylation and mitochondrial co-localization during GLU exposure. (H) Quantitative analysis of AIFM1 (pS116) levels in (G). (I) Effects of RRM2 overexpression combined with Miltefosine on the PI3K/AKT1 pathway, RRM2 expression, and oxeiptosis markers under 50 mM GLU. (J) Quantitative analysis of PI3K/AKT1 pathway activity, RRM2 expression, and oxeiptosis-related protein levels in (I). (K) Apoptosis detection via Annexin V-mCherry/SYTOX Green staining in RRM2-overexpressing cells treated with Miltefosine and 50 mM GLU. (L) Quantitative analysis of Annexin V-mCherry-positive expression from (K). (M) RRM2 overexpression combined with Miltefosine regulates AIFM1 (pS116) expression and mitochondrial co-localization during 50 mM GLU treatment. (N) Quantitative analysis of AIFM1 (pS116) levels in (M). (O) Analysis of the interaction between RRM2 and KEAP1 in HEK293T cells. (P) Protein-protein docking of RRM2 and KEAP1. (Q) Effect of Hydroxyurea (HU) on the interaction between RRM2 and KEAP1. (R) Effect of HU on the expression of oxeiptosis-related proteins promoted by RRM2 overexpression and 50 mM GLU. (S) Quantitative analysis of the related protein levels from (R). (T) Apoptosis detection via Annexin V-mCherry/SYTOX Green staining in RRM2-overexpressing cells treated with HU and 50 mM GLU. (U) Quantitative analysis of Annexin V-mCherry-positive cells from (T). (V) High GLU induced oxeiptosis via the PI3K/AKT1 signaling pathway in an RRM2-dependent manner. Scale bar: 10 µM. All data are presented as mean ± SD, n = 3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Moreover, Z-VAD-FMK (Z-VAD; HY-16658B, MCE, China) was used to inhibit pan-caspase activity, Necrostatin-1 (Nec-1; HY-15760, MCE, China) to inhibit necroptosis, and Hydroxyurea (HU; HY-B0313, MCE, China) to inhibit RRM2 enzymatic activity.

Techniques: Over Expression, Expressing, Activity Assay, Staining, Phospho-proteomics

Journal: Journal of Advanced Research

Article Title: Andrographolide alleviates type 2 diabetic nephropathy through suppressing PI3K/AKT1/RRM2-triggered oxeiptosis

doi: 10.1016/j.jare.2025.10.070

Figure Lengend Snippet: Andrographolide (AND) inhibits hyperglycemia-induced oxeiptosis through dual suppression of PI3K/AKT1 and RRM2 and demonstrates enhanced potential for ameliorating type 2 diabetic nephropathy (T2DN) in combination with metformin (MET). (A) Effects of AND combined with AKT1 overexpression on the PI3K/AKT1 pathway, RRM2 expression, and oxeiptosis-associated proteins under 50 mM glucose (GLU) treatment. (B) Quantitative analysis of PI3K/AKT1 pathway activity, RRM2 expression, and oxeiptosis-related protein levels from (A). (C) Effects of AND combined with si-AKT1 on the PI3K/AKT1 pathway, RRM2 expression, and oxeiptosis markers under 50 mM GLU. (D) Quantitative analysis of PI3K/AKT1 pathway activity, RRM2 expression, and oxeiptosis-related protein levels from (C). (E) Apoptosis detection via Annexin V-mCherry/SYTOX Green staining in cells treated with AND combined with AKT1 overexpression or si-AKT1 under 50 mM GLU. Scale bar: 10 µM. (F) Quantitative analysis of Annexin V-mCherry-positive expression from (E). (G) Effects of AND combined with AKT1 overexpression or si-AKT1 on AIFM1 (pS116) expression and mitochondrial co-localization under 50 mM GLU. Scale bar: 10 µM. (H) Quantitative analysis of AIFM1 (pS116) levels from (G). (I) Effects of AND combined with RRM2 overexpression or si-RRM2 on the PI3K/AKT1 pathway, RRM2 expression, and oxeiptosis markers under 50 mM GLU. (J) Quantitative analysis of PI3K/AKT1 pathway activity, RRM2 expression, and oxeiptosis-related protein levels from (I). (K) Apoptosis detection via Annexin V-mCherry/SYTOX Green staining in cells treated with AND combined with RRM2 overexpression or si-RRM2 under 50 mM GLU. Scale bar: 10 µM. (L) Quantitative analysis of Annexin V-mCherry-positive expression from (K). (M) Effects of AND combined with RRM2 overexpression or si-RRM2 on AIFM1 (pS116) expression and mitochondrial co-localization under 50 mM GLU. Scale bar: 10 µM. (N) Quantitative analysis of AIFM1 (pS116) levels from (M). (O) Schematic diagram illustrating the mechanism by which AND suppresses high glucose-induced oxeiptosis through dual inhibition of PI3K/AKT1 and RRM2. The AND + MET combination provided superior improvement in HOMA-IR (P) and fasting blood glucose levels (Q) compared to MET alone. (R) Representative images of kidney sections subjected to H&E staining (Scale bar: 50 µm; blue arrows indicate cellular vacuolization, green arrows indicate proteinaceous mucus, purple arrows indicate mesangial expansion), MASSON staining (Scale bar: 100 µm; black arrows indicate collagen fiber deposition), PAS staining (Scale bar: 50 µm; blue arrows indicate glycogen deposition), and PASM staining (Scale bar: 50 µm; blue arrows indicate glomerular basement membrane thickening). (S) Effect of the AND + MET combination on the oxeiptosis-related protein expression in renal tissues. (T) Relative quantification of oxeiptosis-related protein levels from (S). (U) Effect of the AND + MET combination on the oxeiptosis-relate protein expression in MDCK cells treated with 50 mM GLU. (V) Relative quantification of oxeiptosis-related protein levels from (U). (W) Apoptosis detection via Annexin V-mCherry/SYTOX Green staining in cells treated with AND combined with MET under 50 mM GLU. Scale bar: 10 µM. (X) Quantitative analysis of Annexin V-mCherry-positive expression from (W). (Y) Effects of AND combined with MET on AIFM1 (pS116) expression and mitochondrial co-localization under 50 mM GLU. Scale bar: 10 µM. (Z) Quantitative analysis of AIFM1 (pS116) levels from (Y). All data are presented as mean ± SD, n = 3. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Moreover, Z-VAD-FMK (Z-VAD; HY-16658B, MCE, China) was used to inhibit pan-caspase activity, Necrostatin-1 (Nec-1; HY-15760, MCE, China) to inhibit necroptosis, and Hydroxyurea (HU; HY-B0313, MCE, China) to inhibit RRM2 enzymatic activity.

Techniques: Over Expression, Expressing, Activity Assay, Staining, Inhibition, Membrane, Quantitative Proteomics

Journal: Frontiers in Neurology

Article Title: PRMT5 inhibition suppresses the PI3K/AKT pathway to attenuate vascular smooth muscle cell pathological phenotype in intracranial aneurysm

doi: 10.3389/fneur.2026.1771196

Figure Lengend Snippet: PRMT5 level was increased in IA models. In vivo analysis of PRMT5 level between the sham and model groups (A) . In vitro analysis of VSMC viability (B) and PRMT5 level (C) among the untreated, Ang II (0.1 μM), Ang II (0.5 μM), and Ang II (1 μM) groups. *: p < 0.05; **: p < 0.01; ***: p < 0.001; ns: not significant.

Article Snippet: EPZ015666 (MCE, China) is an orally bioavailable small-molecule inhibitor of PRMT5 enzymatic activity.

Techniques: In Vivo, In Vitro

Journal: Frontiers in Neurology

Article Title: PRMT5 inhibition suppresses the PI3K/AKT pathway to attenuate vascular smooth muscle cell pathological phenotype in intracranial aneurysm

doi: 10.3389/fneur.2026.1771196

Figure Lengend Snippet: EPZ015666 inhibited PRMT5 enzymatic activity, facilitated transition from synthetic phenotype to contractile phenotype, and suppressed PI3K/AKT pathway in Ang II-treated VSMCs. Comparison of PRMT5, H4R3me2s, α -SMA, OPN, and MMP9 levels among the Model, EPZ (1 μM), EPZ (5 μM), and EPZ (10 μM) groups (A) . Comparison of p-PI3K and p-AKT among the Model, EPZ (1 μM), EPZ (5 μM), and EPZ (10 μM) groups (B) . *: p < 0.05; **: p < 0.01; ***: p < 0.001; ns: not significant.

Article Snippet: EPZ015666 (MCE, China) is an orally bioavailable small-molecule inhibitor of PRMT5 enzymatic activity.

Techniques: Activity Assay, Comparison

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: 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