Journal: Cell Death & Disease

Article Title: FTY720 induces non-canonical phosphatidylserine externalization and cell death in acute myeloid leukemia

doi: 10.1038/s41419-019-2080-5

Figure Lengend Snippet: a MV4-11 cells were treated with 7.5 µM FTY720 or 1 μM RSL-3 (positive control) in the presence or absence of 2 µM Ferrostatin-1 (Fer-1) for 24 h, stained with APC-Ann V and 7-AAD and analyzed by flow cytometry. Mean ± SD, n = 3. Statistical significance for AnnV + 7AAD− (*) and AnnV + 7AAD+ (#) populations was determined by two-way ANOVA followed by Tukey’s multiple comparison test. **** p ≤ 0.0001; * p ≤ 0.05; #### p ≤ 0.0001; # p ≤ 0.05. b MV4-11 cells were treated with 7.5 µM FTY720 or 1.25 mM hydrogen peroxide (H 2 O 2 ; positive control) in the presence or absence of 5 mM N-acetyl-cysteine (NAC) for 22 h, stained with APC-Ann V and 7-AAD and analyzed by flow cytometry. Mean ± SD, n = 3 (DMSO, FTY720); n = 1 (H 2 O 2 ). Statistical significance for AnnV + 7AAD− (*) and AnnV + 7AAD+ (#) populations was determined by two-way ANOVA followed by Tukey’s multiple comparison test; **** p ≤ 0.0001; #### p ≤ 0.0001; ## p ≤ 0.01. c – d CSFE-labeled MV4-11 cells were treated with 7.5 μM FTY720 in the presence or absence of 2 µM Fer-1, 5 mM NAC or E64d/PepA (10 µg/mL each) in medium containing Ann V-AF594 ( c ) or YOYO3 ( d ). Images were obtained using the IncuCyte Live Cell Analysis System and quantified with the Basic Analyzer module. Mean ± SD, n = 3. Note: error bars are included for all data points but may be masked by symbols. c Percent of Ann V-AF594 positive CSFE-labeled cells versus time. d Percent of YOYO3 positive CSFE-labeled cells versus time. e CR-NT or ATG7-deficient MV4-11 cells were treated with 7.5 µM FTY720 or 250 nM ABT-199 (positive control) for 24 h, stained with APC-Ann V and 7-AAD and analyzed by flow cytometry. Mean ± SD, n = 3. Statistical significance for AnnV + 7AAD− (*) and AnnV + 7AAD+ (#) populations was determined by two-way ANOVA followed by Tukey’s multiple comparison test. ns, not significant; **** p ≤ 0.0001; #### p ≤ 0.0001; ## p ≤ 0.01; # p ≤ 0.05. Immunoblot included in inset. f MV4-11 cells were treated with 7.5 µM FTY720 in the presence or absence of E64d/PepA for 24 h and subjected to Ann V/7AAD flow cytometric analysis. Mean ± SD, n = 3. Statistical significance for AnnV + 7AAD− (*) and AnnV + 7AAD+ (#) populations was determined by two-way ANOVA followed by Tukey’s multiple comparison test. ns, not significant; **** p ≤ 0.0001; #### p ≤ 0.0001

Article Snippet: The following chemicals were purchased from the indicated sources: carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (z-VAD-fmk; #HY-16658) from MedChemExpress (Monmouth Junction, NJ, USA), E64d (#S7393) from Selleck Chemicals (Houston, TX, USA), pepstatin A (#260-085) and dynasore (#270-502) from Enzo Life Sciences, Inc. (Farmingdale, NY, USA), and Bafilomycin A1 (#AAJ61835MCR) from Thermo Fisher Scientific.

Techniques: Positive Control, Staining, Flow Cytometry, Comparison, Labeling, Cell Analysis, Western Blot

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: Frontiers in Molecular Neuroscience

Article Title: The Role of Cathepsin B in the Degradation of Aβ and in the Production of Aβ Peptides Starting With Ala2 in Cultured Astrocytes

doi: 10.3389/fnmol.2020.615740

Figure Lengend Snippet: Quantification of the relative amounts of Aβ 2-x (A,B) and of Aβ 1-x (C,D) in conditioned medium of cultured chicken astrocytes after the treatment with E64d (A,C) or CA-074 Me (B,D) over 48 h compared to controls using 1D Urea-SDS-PAGE followed by immunoblot analysis with anti-Aβ 2-x polyclonal antibody (pAb) p77 (A,B) or anti-Aβ 1-x monoclonal antibody (mAb) 82E1 (C,D) . Twenty milliliter (A,B) or 4 ml (C,D) sample volume were used. Aβ 2-40 was significantly reduced after the treatment with E64d (A) or CA-074 Me (B) . No significant reduction of the amounts of Aβ 1-40 or Aβ 1-42 was detected after E64d (C) or CA-074 Me (D) treatment. The labeling of the different Aβ 1-x peptide variants is based on a series of synthetic Aβ (Aβ 1-37, Aβ 1-38, Aβ 1-39, Aβ 1-40, and Aβ 1-42) and their isoelectric point in 2D Urea-SDS-PAGE . A specific Aβ variant, designated as Aβ 1*-x, was effectively reduced by CA-074 Me treatment (D) . Panel (E) shows a section of the amino acid sequences of human and chicken APP, which contains the amino acid sequence of Aβ 1-42 (bold) and adjacent amino acids. In contrast to rodents, no differences in the amino acid sequence exist between humans (NP _958816.1) and chicken (NP _989639.1) in this part of APP (NCBI Reference Sequences are given, http://www.uniprot.org ). Statistics: (A) n = 3, ratio paired T -Test t (2) = 31.43 p < 0.01, (B) n = 3, ratio paired T -Test t (2) = 12.05 p < 0.01, (C) n = 3, Kruskal-Wallis test Aβ 1-37, H (2) = 5. 793, p < 0.05, eta 2 = 0.63, Kruskal-Wallis test Aβ 1-38, H (2) = 3.310, p > 0.05, Kruskal-Wallis test Aβ 1-39, H (2) = 3.310, p > 0.05, Kruskal-Wallis test Aβ 1-40, H (2) = 0.8276, p > 0.05, Kruskal-Wallis test Aβ 1-42, H (2) = 0.1036, p > 0.05, Kruskal-Wallis test Aβ1 * -x, H (2) = 1.471, p > 0.05, (D) n = 5, Kruskal-Wallis test Aβ 1-37 , H (3) = 10.45, p < 0.05, eta 2 = 0.41, Kruskal-Wallis test Aβ 1-38, H (3) = 6.529, p > 0.05, Kruskal-Wallis test Aβ 1-39, H (3) = 8.236, p < 0.05, Kruskal-Wallis test Aβ 1-40, H (3) = 6.101, p > 0.05, Kruskal-Wallis test Aβ 1-42, H (3) = 4.178, p > 0.05, Kruskal-Wallis test Aβ 1*-x, H (3) = 17.23, p < 0.001, eta 2 = 0.79. Dunn's post-hoc test was performed for comparisons to vehicle treated control (con.). Selected comparisons are indicated as follows* p < 0.05; ** p < 0.01, and **** p < 0.0001).

Article Snippet: The cysteine protease inhibitor E64d (100 mM, Peptanova, Sandhausen, Germany), the H + -ATPase inhibitor Bafilomycin A1 (20 μM, Sigma Aldrich, Munich, Germany) the cathepsin B inhibitors CA-074 Me (25 mM, Peptanova), and CA-074 (25 mM, Sigma Aldrich, Munich, Germany) were dissolved in dimethyl sulphoxide (DMSO, Carl Roth, Karlsruhe, Germany) and stored at −20 °C.

Techniques: Cell Culture, SDS Page, Western Blot, Labeling, Variant Assay, Sequencing, Control

Journal: Frontiers in Molecular Neuroscience

Article Title: The Role of Cathepsin B in the Degradation of Aβ and in the Production of Aβ Peptides Starting With Ala2 in Cultured Astrocytes

doi: 10.3389/fnmol.2020.615740

Figure Lengend Snippet: Comparison of the effects of 50 μM CA-074 Me, 50 μM CA-074 and 100 μM E64d on the relative abundances of different Aβ peptide variants in supernatants of H4 cells, H4 APP 751 cells, H4 APP 751 CTSB –/– cells, chicken astrocytes, and human astrocytes compared to controls.

Article Snippet: The cysteine protease inhibitor E64d (100 mM, Peptanova, Sandhausen, Germany), the H + -ATPase inhibitor Bafilomycin A1 (20 μM, Sigma Aldrich, Munich, Germany) the cathepsin B inhibitors CA-074 Me (25 mM, Peptanova), and CA-074 (25 mM, Sigma Aldrich, Munich, Germany) were dissolved in dimethyl sulphoxide (DMSO, Carl Roth, Karlsruhe, Germany) and stored at −20 °C.

Techniques: Comparison