Journal: Journal of Nanobiotechnology

Article Title: TROP2-directed nanobody-drug conjugate elicited potent antitumor effect in pancreatic cancer

doi: 10.1186/s12951-023-02183-9

Figure Lengend Snippet: Endocytosis and in vitro antitumor activity of HuNb TROP2-HSA -MMAE. A The process of HuNb TROP2-HSA -MMAE internalization into BxPC-3 cells. Blue: nuclei stained by Hoechst33342; green: HuNb TROP2-HSA -MMAE labeled with Alexa Fluor™ 488. B HuNb TROP2-HSA -MMAE underwent lysosomal degradation after internalization. Blue: nuclei; green: HuNb TROP2-HSA -MMAE; red: lysosomes. C The expression of TROP2 on the surface of three human pancreatic cell lines was determined by flow cytometry and the cytotoxic effects of HuNb TROP2-HSA -MMAE treatment for 2 or 3 days on these cancer cells were evaluated by MTT assay

Article Snippet: In the drug release assays, 0.5 mg/mL HuNb TROP2-HSA -MMAE was incubated with 5 μg/mL cathepsin B (MCE, HY-P78682) at pH 6.0 for different time.

Techniques: In Vitro, Activity Assay, Staining, Labeling, Expressing, Flow Cytometry, MTT Assay

Journal: Journal of Nanobiotechnology

Article Title: TROP2-directed nanobody-drug conjugate elicited potent antitumor effect in pancreatic cancer

doi: 10.1186/s12951-023-02183-9

Figure Lengend Snippet: Cell apoptosis induced by HuNb TROP2-HSA -MMAE. A The effect of HuNb TROP2-HSA -MMAE on apoptosis of BxPC-3 and PK-59 cells was detected by Annexin V-FITC/PI staining. Annexin V + PI − (Q4) represents early apoptotic cells and Annexin V + PI + (Q2) represents late apoptotic cells. B A statistical analysis of three replicate experiments of ( A ). The proportion of apoptotic cells is the sum of Q2 and Q4. C Western Blot demonstrated the expressions of apoptosis-related proteins in pancreatic cancer cells after 72 h of treatment of HuNb TROP2-HSA -MMAE

Article Snippet: In the drug release assays, 0.5 mg/mL HuNb TROP2-HSA -MMAE was incubated with 5 μg/mL cathepsin B (MCE, HY-P78682) at pH 6.0 for different time.

Techniques: Staining, Western Blot

Journal: Journal of Nanobiotechnology

Article Title: TROP2-directed nanobody-drug conjugate elicited potent antitumor effect in pancreatic cancer

doi: 10.1186/s12951-023-02183-9

Figure Lengend Snippet: In vivo distribution and metabolism of HuNb TROP2-HSA -MMAE. A Living imaging after intravenous injection of 200 μg Cy7-HuNb TROP2-HSA -MMAE for different times. B Fluorescence distribution in major organs at 24 h post-injection. C Quantitative analysis of the fluorescence intensity in ( B )

Article Snippet: In the drug release assays, 0.5 mg/mL HuNb TROP2-HSA -MMAE was incubated with 5 μg/mL cathepsin B (MCE, HY-P78682) at pH 6.0 for different time.

Techniques: In Vivo, Imaging, Injection, Fluorescence

Journal: Journal of Nanobiotechnology

Article Title: TROP2-directed nanobody-drug conjugate elicited potent antitumor effect in pancreatic cancer

doi: 10.1186/s12951-023-02183-9

Figure Lengend Snippet: In vivo antitumor activity of HuNb TROP2-HSA -MMAE. A Tumor volume during 4 weeks of treatment with different doses of HuNb TROP2-HSA -MMAE. B Survival curves of mice receiving various treatments. C Each line represents the tumor volume for an individual mouse. D H&E staining and IHC for Ki-67, cleaved caspase-3 and cleaved PARP of tumor tissues

Article Snippet: In the drug release assays, 0.5 mg/mL HuNb TROP2-HSA -MMAE was incubated with 5 μg/mL cathepsin B (MCE, HY-P78682) at pH 6.0 for different time.

Techniques: In Vivo, Activity Assay, Staining

Journal: Advanced Science

Article Title: Deoxynivalenol Exposure Leads to Abnormal Renal Tubular Autophagy Flow

doi: 10.1002/advs.202505486

Figure Lengend Snippet: DON exposure induced TCMK‐1 lysosomal dysfunction. A, B) TCMK‐1 were treated with 200 ng mL −1 DON for 48 h, followed by replacing the medium containing MitoTempo (10 µM) for 24 h. 4HNE levels were detected by immunoblotting with antibodies. C, D) Stained TCMK‐1 with FerroOrange was observed by confocal microscopy (Scale bar = 10 µm). E) Heatmap of 23 proteins associated with the lysosome in the H‐DON groups compared to the control groups (n = 3 technical replicates). F) The representative TEM images of TCMK‐1 (Scale bar = 500 nm). The blue area was the lysosome. G, H) LAMP1, LAMP2, CTSB, and CTSD levels were detected by immunoblotting with antibodies. I–K) Representative images of TCMK‐1 transfected Ad‐mCherry‐GFP‐LC3B in the presence of DON or MitoTempo treatment. The number of green‐red positive puncta in each cell was determined, and the ratio of autophagosomes to autolysosomes was calculated. The values were expressed as the mean ± S.D. ( *** P < 0.001 and **** P < 0.0001 compared with the control group).

Article Snippet: The following primary antibodies were utilized: GAPDH (Servicebio, GB15004‐100; 1:2000), 4HNE (ABclonal, A24456 ; 1:5000), KIM‐1 (ABclonal, A2831; 1:1000), SLC3A2 (ABclonal, A24735; 1:2000), FTL (Proteintech, 10727‐1‐AP; 1:5000), ATG4B (ABclonal, A5059; 1:2000), LC3B (Proteintech, 23274‐1‐AP; 1:1000), TOMM20 (Wanlei, WL02512; 1:1000), VDAC3 (ABclonal, A26244; 1:1000), LAMP1 (ABclonal, A21194; 1:10 000), LAMP2 (ABclonal, A1961; 1:3000), CTSB (ABclonal, A19005; 1:1000), CTSD (ABclonal, A19680; 1:4000).

Techniques: Western Blot, Staining, Confocal Microscopy, Control, Transfection

Journal: Advanced Science

Article Title: Deoxynivalenol Exposure Leads to Abnormal Renal Tubular Autophagy Flow

doi: 10.1002/advs.202505486

Figure Lengend Snippet: DON exposure induced renal lysosomal dysfunction in mice. A–C) Changes in LC3 and ATG4B protein levels in the kidney were detected by western blotting. D–H) Changes in LAMP1, LAMP2, CTSB, and CTSD protein levels in the kidney were detected by western blotting. D, K) IHC staining for LAMP2 in the kidney (Scale bar = 200 µm). J, L) IHC staining for CTSD in the kidney (Scale bar = 200 µm). O) The representative TEM images of the kidney (Scale bar = 500 nm). The blue area was the lysosome. M) Correlation between the density of LAMP2 immunostaining and Scr in the mouse kidney. N) Correlation between density of CTSD immunostaining and Scr in the mouse kidney. The values were expressed as the mean ± S.D. ( * P < 0.05, *** P < 0.001, and **** P < 0.0001 compared with the control group).

Article Snippet: The following primary antibodies were utilized: GAPDH (Servicebio, GB15004‐100; 1:2000), 4HNE (ABclonal, A24456 ; 1:5000), KIM‐1 (ABclonal, A2831; 1:1000), SLC3A2 (ABclonal, A24735; 1:2000), FTL (Proteintech, 10727‐1‐AP; 1:5000), ATG4B (ABclonal, A5059; 1:2000), LC3B (Proteintech, 23274‐1‐AP; 1:1000), TOMM20 (Wanlei, WL02512; 1:1000), VDAC3 (ABclonal, A26244; 1:1000), LAMP1 (ABclonal, A21194; 1:10 000), LAMP2 (ABclonal, A1961; 1:3000), CTSB (ABclonal, A19005; 1:1000), CTSD (ABclonal, A19680; 1:4000).

Techniques: Western Blot, Immunohistochemistry, Immunostaining, Control

Journal: EBioMedicine

Article Title: Proteolytic cleavage of amyloid precursor protein by ADAM10 mediates proliferation and migration in breast cancer

doi: 10.1016/j.ebiom.2018.11.012

Figure Lengend Snippet: Expression of APP and its proteolytic product in breast cancer cell lines. (A) Immunoblot analysis using an antibody against APP N-terminal (clone 22C11) showing differential expression of APP on breast cancer cell lines BT-549, MDA-MB231, MCF7, MDA-MB468, SK-BR-3 and ZR-75-1. α-tubulin was used as a control. Densitometric analysis of APP expression from triplicate experiments was shown at the lower panel. (mean ± SD; n = 3). (B) Conditioned medium from cell line culture was examined using antibodies specific for sAPPα and sAPPβ, in addition to clone 22C11. Secretion of sAPPα, but not sAPPβ into cell culture supernatant can be detected. All experiments were performed at least three times.

Article Snippet: The mammalian expression constructs for APP (pCAX-APP751) and sAPPα (pCAX-APPs-751-alpha) were obtained from Addgene.

Techniques: Expressing, Western Blot, Quantitative Proteomics, Control, Cell Culture

Journal: EBioMedicine

Article Title: Proteolytic cleavage of amyloid precursor protein by ADAM10 mediates proliferation and migration in breast cancer

doi: 10.1016/j.ebiom.2018.11.012

Figure Lengend Snippet: Overexpression of full length APP and sAPPα enhanced breast cancer proliferation and migration. (A) Expression of full length or sAPPα promoted proliferation in breast cancer cell line (MDA-MB231). MDA-MB231 was transfected with full length (FL), sAPP or control (CTR) construct and were seeded (1 × 10 3 ) in a 96 well plate. Western blotting analysis for APP expression after transfection was shown (insert). Cell proliferation was measured at day 4 by CCK8 assay. (mean ± SD; n = 3). Statistical analysis was performed using student t -test. ***p < 0.001. (B) APP overexpression enhanced migration of breast cancer. Cells transfected with different APP constructs (1 × 10 5 ) were seeded with 0.2 ml serum free medium in the upper chamber of Boyen chamber after overnight serum starvation. 0.5 ml medium supplemented with 10% FBS was added to the lower chamber. Migration was evaluated for 22 h. (mean ± SD; n = 3). Statistical analysis was performed using student t-test. ***p < 0.001. All experiments were performed at least three times.

Article Snippet: The mammalian expression constructs for APP (pCAX-APP751) and sAPPα (pCAX-APPs-751-alpha) were obtained from Addgene.

Techniques: Over Expression, Migration, Expressing, Transfection, Control, Construct, Western Blot, CCK-8 Assay

Journal: EBioMedicine

Article Title: Proteolytic cleavage of amyloid precursor protein by ADAM10 mediates proliferation and migration in breast cancer

doi: 10.1016/j.ebiom.2018.11.012

Figure Lengend Snippet: ADAM10 and ADAM17 in APP processing of breast cancer cell lines. (A) Immunoblot analysis using antibodies against ADAM10 and ADAM17 showed the expression of both proteins in breast cancer cell lines (BT-549, MDA-MB231, MCF7, MDA-MB468, SK-BR-3 and ZR-75-1). β-actin was used as a control. Densitometric analysis of protein expression on the predominant form detected (pro-ADAM17 and mature ADAM10) from replicate experiments were shown at the lower panel. (mean ± SD; n = 3). (B) Knockdown of ADAM 10 but not ADAM17 showed reduced sAPP level in conditioned medium from cell line culture. Breast cancer cell line (MDA-MB231) was transfected with ADAM10 or ADAM17 siRNA showed a marked reduction in protein and mRNA expression. A decreased level of sAPP in the conditioned supernatant was found with ADAM10 but not ADAM17 knockdown. Densitometric analysis of APP and sAPP was shown at the right panel. (mean ± SD; n = 3). Statistical analysis was performed using student t -test. ***p < 0.001. All experiments were performed at least three times.

Article Snippet: The mammalian expression constructs for APP (pCAX-APP751) and sAPPα (pCAX-APPs-751-alpha) were obtained from Addgene.

Techniques: Western Blot, Expressing, Control, Knockdown, Transfection