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: Cell Biology International

Article Title: Modulation of Autophagy by Ursolic and Betulinic Acids: Distinct Cytotoxic and Membrane‐Disruption in Malignant and Nonmalignant Cells

doi: 10.1002/cbin.70073

Figure Lengend Snippet: UA induces lysosomal membrane permeabilization and cell death in HaCaT cells. We treated HaCaT with 20 µM triterpenoids or 0.25% (v/v) DMSO for 6 h and (a) quantified total cytosolic CTSB by ELISA assay and (b) immunostaining for CTSB (green) and lysosomes loaded with LysoTracker Red DND‐99 (red). At right, fluorescence plot profiles represented line scans of CTSB and LTR‐loaded lysosomes. (c) After 24‐h treatment with 0.25% (v/v) DMSO and 20 µM triterpenoids, cells were stained with annexin V and propidium iodide and gated according to (FL1) and (FL3) fluorescence following FACS. A representative scatterplot displays the subpopulations Q1, Q2, Q3, and Q4 (upper panel), while a graph illustrates the frequency distribution of these subpopulations (bottom panel). (d) Gated AV − /PI + (Q3) cells plotted according to 90° Side scatter (SSC) and forward low‐angle light scatter (FSC) parameters and analyzed through scatterplot distribution (upper panel). We represented the frequency of cell subpopulations with bars (bottom panel). (e) Activity of CTSB and CTSL in lysosomal fraction by fluorometric assay after treatment with 20 µM triterpenoids or 0.25% (v/v) DMSO at indicative times. (f) After a 48‐h recovery period, HaCaT cells treated with 0.50% (v/v) DMSO or 40 µM triterpenoids were stained with acridine orange (AO) and imaged. At the bottom, fluorescence plot profiles represented line scans of the lysosomotropic dye AO's green and red fluorescence intensity. Results are from at least three replicates and two independent experiments, presented as mean ± standard error. ANOVA post hoc test Dunnett T3 (b, d‐Q4, and e) or Bonferroni (c and d‐Q1–Q3) were performed, and significance levels are indicated as * p < 0.05, ** p < 0.01, *** p < 0.001. Asterisks above bars represented the statistical significance compared to the DMSO control. Scale bars: 10 μm (a and f).

Article Snippet: Following treatment of HaCaT cells with 20 μM triterpenoids or DMSO (0.25% v/v) for 6 h, we assessed the quantity of cytosolic CTSB using an ELISA assay (R&D Systems DY2176).

Techniques: Membrane, Enzyme-linked Immunosorbent Assay, Immunostaining, Fluorescence, Staining, Activity Assay, Control

Journal: Cell death & disease

Article Title: Galectin-3 promotes secretion of proteases that decrease epithelium integrity in human colon cancer cells.

doi: 10.1038/s41419-023-05789-x

Figure Lengend Snippet: Fig. 7 Galectin-3 expression induces activation of PYK2, STAT1 and GSK3α/β signalling. Expression of 37 protein kinases in SW620 cells in response to 10 µg/ml galectin-3 or BSA for 0.5 h was assessed by Proteome Profiler Human Phospho-Kinase Array (A, Percentage changes of the kinases in cell response to galectin-3 in comparison to control are shown at the bottom panel). The presence of galectin-3 increases the phosphorylation of PYK2, GSK3α/β, and STAT1 and decreases phosphorylation of STAT3. SW620 cells treated with 10 µg/ml galectin-3 for different times were assessed by immunoblotting using antibodies against p-PYK2, p-STAT-1, p-GSK3α/β or p-STAT-3 (B). The blots were striped and reprobed with antibodies against PYK2, STAT-1, GSK3α/β or STAT-3. The band density was quantified and expressed as percentages of phospho-/non-phosphorylated proteins (C). In D and E, SW620 cells were treated with 10 µg/ml galectin-3 or BSA followed by introduction of GSK3α/β inhibitor SB 216763 (SB) or PKY2 inhibitor PF-431396 (PF) for 15 min and the levels of phosphorylated PYK2, STAT-1, GSK3α/β or STAT-3 were analysed by immunoblotting. The blots were striped and reprobed with antibodies against PYK2, STAT-1, GSK3α/β or STAT-3. The densities of the blots from three independent experiments were quantified and are expressed as the percentage of phosphorylated/non-phosphorylated levels of each protein. ***P < 0.001, **P < 0.01, *P < 0.05 (ANOVA).

Article Snippet: Biotinylated-anti-galectin-3 (BAF1154) antibody and antibodies against galectin-3 (MAB1154), STAT1 (MAB1490), Phospho-STAT1(MAB2894), STAT3 (MAB1799), Phospho-STAT3 (MAB4934), PYK2 (AF4589), phosphorPYK2 (MAB6210), GSK3α/β (AF2157), Phospho-GSK3α/β (AF1590); Proteome Profiler Human Protease Array (ARY021B), Proteome Profiler Human Phospho-Kinase Array (ARY003C); Human MMP-13 DuoSet ELISA (DY511), Human Cathepsin-B DuoSet ELISA (DY2176) PYK2 inhibitor PF-431396 (4278) and GSK3α/β inhibitor SB 216763 (1616/1) were all purchased from R&D Systems (Abingdon, UK).

Techniques: Expressing, Activation Assay, Comparison, Control, Phospho-proteomics, Western Blot

Journal: Cell death & disease

Article Title: Galectin-3 promotes secretion of proteases that decrease epithelium integrity in human colon cancer cells.

doi: 10.1038/s41419-023-05789-x

Figure Lengend Snippet: Fig. 8 Galectin-3 induces CTSB secretion through PYK2-GSK3α/β activation. SW620 (A) and HCT116 (B) cells were treated with 10 µg/ml galectin-3 or BSA without or with SB216763 (SB), PF431396 (PF) or DMSO overnight. The concentrations of cathepsin-B in the supernatants were analysed by ELISA. Data are presented as mean ± SEM of three independent experiments. ***P < 0.001, **P < 0.01, *P < 0.05 (ANOVA).

Article Snippet: Biotinylated-anti-galectin-3 (BAF1154) antibody and antibodies against galectin-3 (MAB1154), STAT1 (MAB1490), Phospho-STAT1(MAB2894), STAT3 (MAB1799), Phospho-STAT3 (MAB4934), PYK2 (AF4589), phosphorPYK2 (MAB6210), GSK3α/β (AF2157), Phospho-GSK3α/β (AF1590); Proteome Profiler Human Protease Array (ARY021B), Proteome Profiler Human Phospho-Kinase Array (ARY003C); Human MMP-13 DuoSet ELISA (DY511), Human Cathepsin-B DuoSet ELISA (DY2176) PYK2 inhibitor PF-431396 (4278) and GSK3α/β inhibitor SB 216763 (1616/1) were all purchased from R&D Systems (Abingdon, UK).

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay