Journal:

Article Title: Imaging Proteolysis by Living Human Breast Cancer Cells 1

doi:

Figure Lengend Snippet: Protease Inhibitors Reduce Degradation of DQ-Collagen IV by BT20 Cells and BT549 Cells.

Article Snippet: In studies with inhibitors, the cells were plated as above and grown in the presence of 100 nM of cystatin C (M. Abrahamson, University of Lund, Sweden), an endogenous inhibitor of cysteine proteases, 10 μ M of the highly selective cathepsin B inhibitor CAO74 or its membrane-permeable derivative CA074Me (Peptides International), 10 μ M of the broad spectrum cysteine protease inhibitor E-64 (Sigma) or its membrane-permeable derivative E-64d (Peptides International), 10 μ M of a broad spectrum MMP inhibitor (BB3103; a kind gift of British Biotech), 1 μ M of the serine protease inhibitor aprotinin (Sigma), or 5 μ g/ml of cytochalasin B (Sigma) over the entire period of observation.

Techniques: Control

Journal:

Article Title: Imaging Proteolysis by Living Human Breast Cancer Cells 1

doi:

Figure Lengend Snippet: Inhibitors of cysteine proteases and cathepsin B reduced intracellular degradation of DQ-collagen IV by living BT549 human breast cancer cells. Phase images of living BT549 cells are superimposed on optical sections through the cells. Green fluorescent products resulting from cleavage of DQ-collagen IV can be observed in the control cells and those treated with BB3103 (10 µM). A striking decrease in green fluorescent products can be observed in cells treated with E-64d (10 µM) or CA074Me (10 µM).

Article Snippet: In studies with inhibitors, the cells were plated as above and grown in the presence of 100 nM of cystatin C (M. Abrahamson, University of Lund, Sweden), an endogenous inhibitor of cysteine proteases, 10 μ M of the highly selective cathepsin B inhibitor CAO74 or its membrane-permeable derivative CA074Me (Peptides International), 10 μ M of the broad spectrum cysteine protease inhibitor E-64 (Sigma) or its membrane-permeable derivative E-64d (Peptides International), 10 μ M of a broad spectrum MMP inhibitor (BB3103; a kind gift of British Biotech), 1 μ M of the serine protease inhibitor aprotinin (Sigma), or 5 μ g/ml of cytochalasin B (Sigma) over the entire period of observation.

Techniques: Control

Journal: The FEBS journal

Article Title: Verapamil treatment induces cytoprotective autophagy by modulating cellular metabolism.

doi: 10.1111/febs.14064

Figure Lengend Snippet: Fig. 7: Verapamil induces autophagic flux Western blots for LC3 protein levels after treatment with increasing concentrations of Verapamil (Ver; 50, 100, 200 µM; 6 h) alone or in combination with lysosomal protease inhibitors – E64d/Pepstatin A (E/PepA; 10 µM; 6 h) (A-E). Actin or ERK2 were used as a loading control. Representative images of the Western blots are shown (upper panels) together with the quantification of LC3-II fold increase in comparison to control, untreated cells (lower panels). Each performed experiment is presented as an individual data point set with mean (±SEM), n= 3 to 6.

Article Snippet: Verapamil chloride (Sigma-Aldrich, V4629), E64d (LKT Laboratories, E0003), Pepstatin A (Calbiochem, 516481), Chloroquine (Sigma-Aldrich, C6620), Sodium oxamate (Sigma-Aldrich, O2751).

Techniques: Western Blot, Control, Comparison

Journal: American Journal of Cancer Research

Article Title: Endogenous S100P-mediated autophagy regulates the chemosensitivity of leukemia cells through the p53/AMPK/mTOR pathway

doi: 10.62347/NWXE8730

Figure Lengend Snippet: S100P knockdown increased HBSS-induced autophagy in leukemia cells. A. HL-60 and Jurkat cells were transfected with S100P shRNA or control shRNA and then starved by HBSS for 2 h. LC3-I/II and p62 levels were assayed by western blot; B. HL-60 and Jurkat cells were transfected with S100P shRNA or control shRNA and then pre-treated for 1 h with pepstatin A (PA, 10 μM) and E64d (10 μM) as indicated. Cells were subsequently treated for 2 h with HBSS in continuous presence or absence PA/E64d inhibitors. LC3-I/II and p62 levels were assayed by western blot (n=3, *P<0.05 vs. shS100P group); C. Ultrastructural features in HL-60 and Jurkat cells transfected with S100P shRNA or control shRNA after 2 h treatment of HBSS. More autophagolysosomes were seen in S100P shRNA plus HBSS-treated cells than in cells treated with control shRNA plus HBSS. Red arrows indicated autophagolysosomes. Scale bar: 500 nm; D. HL-60 and Jurkat cells were transfected with S100P shRNA or control shRNA and then starved by HBSS for 2 h and then immunostained with LC3B-specifc antibody (red) and DAPI (blue). Scale bar: 10 μm.

Article Snippet: Reagents and cell culture 3-Methyladenine (3-MA), Cytarabine (Ara-C), adriamycin (ADM), Tenovin-6, Compound C and lysosomal protease inhibitors E64d, pepstatin A [ 22 ] were purchased from GlpBio (Montclair, CA, USA); AML cell lines (MV4-11, THP-1, HL-60, KASUMI1) and ALL cell lines (NALM-6, MOLT4, Jurkat) from Xiangya School of Medicine Type Culture Collection (Changsha, China).

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

Journal: Science (New York, N.Y.)

Article Title: Triggered recruitment of ESCRT machinery promotes endolysosomal repair

doi: 10.1126/science.aar5078

Figure Lengend Snippet: (A) HeLa cells treated with or without LLOME were costained for CHMP4A and GAL3. (B and C) U2OS cells treated as above were costained for ALIX and the indicated ESCRT-III proteins. (D) U2OS cells preincubated for 5 min in the absence or presence of the cathepsin inhibitor E64d were exposed to LLOME and costained for CHMP4A and ALIX. (E) LLOME-treated U2OS cells were stained for CHMP4A and EEA1 or LAMP1. Fractional overlap between CHMP4A puncta and labeled compartments is shown at right (n = 7 cells for EEA1, 13 cells for LAMP1). (F) U2OS cells or (G) HeLa cells producing CHMP4C-GFP were treated with LLOME and immunolabeled as indicated before processing for deep-etch electron microscopy. Top panels depict two-dimensional views with pseudocolored immunogold; bottom panels show corresponding anaglyphs, to be viewed with dual color glasses. In all fluorescence micrographs, representative cells are shown outlined by dashed white lines; boxed areas are magnified at right; and coincidence of green and magenta appears white. Scale bars equal 10 µm (A to E; 2 µm in magnified views); 100 nm (F and G).

Article Snippet: Compounds were used at the following concentrations unless explicitly stated: 25 µM BAPTA-AM (no. 15551; Cayman Chemical, Ann Arbor, MI, USA); 200 µM E64d (no. 13533; Cayman Chemical); 200 µM GPN (no. sc-252858; Santa Cruz Biotechnology Dallas, TX, USA); 1 mM LLOME (no. L7393; Sigma-Aldrich); and 10 µM wortmannin (no. W1628; Sigma-Aldrich).

Techniques: Staining, Labeling, Immunolabeling, Electron Microscopy, Fluorescence