Journal: Cell Death & Disease
Article Title: Ceramide synthase-6 confers resistance to chemotherapy by binding to CD95/Fas in T-cell acute lymphoblastic leukemia
Figure Lengend Snippet: CERS6 is overexpressed in ALL. a Biosynthesis and metabolism of ceramides. Ceramides are generated either de novo or via the salvage pathway. They are metabolized to sphingosine or may serve as substrates for the synthesis of glucosyl ceramides, lactosyl ceramides or sphingomyelins. Sphingomyelins degrade to ceramides via the sphingomyelinase pathway and are synthesized from ceramides via sphingomyelin synthase. b mRNA expression of the six ceramide synthase isoforms in the NCI PPTP panel of 23 cell lines comprising six different pediatric cancers. RBD rhabdomyosarcoma, BT brain tumor, EFT Ewing’s family of tumors, NB neuroblastoma, ALL acute lymphoblastic leukemia, LYM lymphoma. The mRNA expression was determined within the NCI Pediatric Preclinical Testing Program. c CERS6 protein expression in acute lymphoblastic leukemia (ALL) cell lines in comparison to peripheral blood mononuclear cells (PBMCs) and T lymphocytes obtained from blood of healthy human volunteers. GAPDH was used as a loading control. d C 16 -Ceramides in T-cell ALL cells in comparison to PBMCs and T lymphocytes. Ceramide levels were quantitated by HPLC/MS/MS (PBMC: 0.28 ± 0.04, n = 18; T lymphocytes: 0.36 ± 0.01, n = 3; T-ALL: 0.54 ± 0.09, n = 9). e CERS6 protein expression in T lymphocytes isolated from primary lymphoid malignancy samples in comparison to normal T lymphocytes *** p
Article Snippet: Chemicals and reagents Sphingolipid standards includingC14:0-, C16:0-, C17:0-, C18:0-, C18:1-, C20:0-, C24:0-, C24:1-ceramide and C18:0-, C18:1-, C24:0-, C24:1-dihydroceramide were purchased from Avanti Polar Lipids (Alabaster, AL); ammonium formate and formic acid were obtained from Fisher Scientific (Pittsburg, PA); chloroform, ethyl acetate, methanol, 2-propanol, NaF, NaHCO3 , Na3 VO4 , Tris-HCl, Triton X-100, pepstatin A, aprotinin, leupeptin, 200 proof ethanol, isopropanol, puromycin, dexamethasone, and anti-FLAG-M2 (1 μg/ml) antibody from Sigma-Aldrich (St. Louis, MO); ABT-737 from Cayman Chemical (Ann Arbor, MI); DTT, EDTA, NaCl, PMSF, SDS, TBE, trypsin/EDTA, Lipofectamine®, PLUSTM reagent, Superscript® III first-strand synthesis system for RT-PCR from Thermo Fisher Scientific (Waltham, MA); Triton X-114 from Acros Organics (Morris, NJ); Z-IETD from R & D Systems (Minneapolis, MN); Fas ligand from GeneTex (Irvine, CA); anti-CERS6, anti-FLIP and anti-GAPDH antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); anti-Fas and anti-FADD from BD Transduction Laboratories (San Jose, CA); anti-HA antibody from Roche (Indianapolis, IN); anti-caspase-3, anti-cleaved-caspase-3, anti-caspase-8 and anti-PARP from Cell Signaling Technology (Danvers, MA); Age 1-HF, Bam H1-HF, Eco R1-HF, Mlu 1-HF, Pme 1 and Sgf 1 restriction enzymes from New England Biolabs (Ipswich, MA); bovine serum albumin from Jackson ImmunoResearch Laboratories (West Grove, PA); all oligos were synthesized from Integrated DNA Technologies (Coralville, IA).
Techniques: Generated, Synthesized, Expressing, High Performance Liquid Chromatography, Mass Spectrometry, Isolation