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ATCC
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Syngenta
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Syngenta
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Merck & Co
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Mcule Inc
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Shanghai Macklin Biochemical
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Novartis
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Journal: Cell Death & Disease
Article Title: An increase of lysosomes through EGF-triggered endocytosis attenuated zinc-mediated lysosomal membrane permeabilization and neuronal cell death
doi: 10.1038/s41419-024-07192-6
Figure Lengend Snippet: A Bars represent LDH release at 20 hours after exposure to zinc (40 µM), with or without EGF (100 ng/ml), methyl-beta-cyclodextrin (MβCD, 2 mM; caveolin-dependent endocytosis inhibitor), chlorpromazine (CP, 2 µM; clathrin-dependent endocytosis inhibitor), compound 56 (Cpd56, 1 µM; inhibitor of EGFR tyrosine kinase activity), or ciliobrevin A (ciliob, 500 nM; dynein inhibitor) (mean ± SEM, n = 3-15 taken from ≥3 independent biological replicate experiments, # p < 0.05, ## p < 0.01, or not significant (ns) for zinc, and ** p < 0.01 or *** p < 0.001 for Zn + EGF, analyzed using ANOVA with Dunnett’s post-hoc test). B Western blot analysis (upper) and quantitative graphs (lower) demonstrate the protein levels of EGFR and LAMP-1 at 0.5 hours after EGF (100 ng/ml) treatment with or without endocytosis inhibitors: MβCD (2 mM), CP (2 µM), Cpd56 (1 µM), or Ciliob (500 nM). The bar graphs indicate the band density ratio of EGFR or LAMP-1 to Actin (mean ± SEM, n = 4–8 independent biological replicate experiments, # p < 0.05, ## p < 0.01, or ### p < 0.001 for control , and * p < 0.05 or ** p < 0.01 for EGF, analyzed by ANOVA using Dunnett’s post-hoc test). C Microscopic images (left) and quantitative graphs (right) show LTR-labeled neuronal cells at 1 h after EGF (100 ng/ml) treatment, with or without endocytosis inhibitors: MβCD (2 mM), CP (2 µM), Cpd56 (1 µM), or Ciliob (500 nM). Scale bar: 100 µm. The bar graphs represent LTR intensity measured using the Image J program (mean ± SEM, n = 9 or 15 different fields taken from ≥4 independent biological replicate experiments, ## p < 0.01 or #### p < 0.0001 for control, and ** p < 0.01 for EGF, analyzed by ANOVA with Dunnett’s post-hoc test). D Western blot analysis demonstrates the release of cathepsin B (CTSB) from lysosomes to the cytosol. Cytosolic proteins were extracted at 6 h after exposure to 40 µM zinc with or without EGF (100 ng/ml), MβCD (2 mM), CP (2 µM), Cpd56 (1 µM), or Ciliob (500 nM). The lower bar graph presents the band intensity ratio of mature CTSB to Actin (mean ± SEM, n = 6 or 3 independent biological replicate experiments, # p < 0.05, or #### p < 0.0001 for zinc, and * p < 0.05, ** p < 0.01, or **** p < 0.0001 for Zn + EGF, analyzed by ANOVA using Dunnett’s post-hoc test).
Article Snippet: To inhibit the endocytosis of EGF receptor (EGFR), 2 mM methyl-beta-cyclodextrin (MβCD; Sigma-Aldrich), 2 µM chlorpromazine (CP; Sigma-Aldrich), 1 µM
Techniques: Activity Assay, Western Blot, Control, Labeling