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: Clinical Cancer Research

Article Title: Discovery and Targeting of a Noncanonical Mechanism of Sarcoma Resistance to ADI-PEG20 Mediated by the Microenvironment

doi: 10.1158/1078-0432.CCR-22-2642

Figure Lengend Snippet: Ass1 KO tumor cells macropinocytose EVs to overcome arginine deprivation. A, Fold change in number of live 1037 cells after 96 hours of treatment with ADI-PEG20 with and without both cocultured MEFs and 25 μmol/L EST. See also Supplementary Fig. S5D. B, Fold change in number of live 1037 cells after 96 hours of treatment with ADI-PEG20 with and without both cocultured MEFs and 25 μmol/L imipramine. C, Areas under curves of proliferation of 1037 cells treated with ADI-PEG20 and various concentrations of EIPA and imipramine in co-culture with MEFs. D, Areas under curves of proliferation of MEFs treated with ADI-PEG20 and various concentrations of EIPA and imipramine in coculture with 1037 cells. E, Uptake of MEF EVs by 1037 cells with or without 20 μmol/L imipramine during ADI-PEG20 treatment. More fluorescence remaining in media indicates less uptake. F, Heavy arginine labeling intensity of 1037 cells cocultured for 24 hours with MEFs that were either unlabeled or fully labeled with 13 C 15 N L-arginine, with and without 20 μmol/L imipramine. G, Growth of BVMA01R tumors grafted into syngeneic C57BL/6J mice with and without both ADI-PEG20 and imipramine treatments. H, Weights of mice from G . I, Quantification of ASS1 levels in harvested tumors from G . Data are mean ± SD except in G and H (mean ± SEM) [ n = 3 in A – E ; n = 2 in F ; n = 5–12 in G and H ; n = 3–6 in I ]. Two-way ANOVA tests for G and H . Two-tailed paired t tests for A, B, E . One-tailed unpaired t test for F . Two-tailed unpaired t tests for I .

Article Snippet: EST (aloxistatin; MedChemExpress), imipramine (Millipore Sigma), 5-(N-ethyl-N-isopropyl)-amiloride (EIPA; Millipore Sigma), chloroquine (Millipore Sigma), tonabersat (Cayman Chemical Company), carbenoxolone (Apexbio Technology), Pitstop 2 (Millipore Sigma), docetaxel (Millipore Sigma), and gemcitabine (Millipore Sigma) were administered at the indicated concentrations, and controls were treated with the same volume of vehicle.

Techniques: Co-Culture Assay, Fluorescence, Labeling, Two Tailed Test, One-tailed Test

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