C2 dihydroceramide

Reagents and chemicals: C₆-, C₈-, C₁₆-, C₁₈-, C₂₄-ceramide, C₂-dihydroceramide and SM were obtained from Sigma-Aldrich, Alexis (Grünberg, Germany) or Acros (Geel, Belgium), and DECCA and water-free toluene (over molecular sieve 4 A°) were obtained from Fluka (Taufkirchen, Germany). Methanol, ethyl acetate, cyclohexane, acetonitrile and iodine were supplied from Merck (Darmstadt, Germany), chloroform by J.T. Baker (Deventer, Netherlands). All chemicals were of reagent grade. Solvents for HPLC were either freshly distilled or commercially available HPLC grade.

Desmethylimipramine (desipramine), N,N′-Bis[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]-3,3′-p-phenylene-bis-acrylamide dihydrochloride (GW4869), SMase from Staphylococcus aureus, C2 dihydroceramide, S1P, and BafA1 were purchased from Sigma-Aldrich (Bornem, Belgium), C2-ceramide from Santa Cruz Biotechnology (Boechout, Belgium) and C6-ceramide from Enzo Life Sciences (Antwerp, Belgium). Human recombinant TNFα was kindly provided by Pr. Athanase Visvikis (Université de Lorraine, Nancy, France). Stem cell factor (SCF) and interleukin (IL)-3 were purchased from ReliaTech (Wolfenbüttel, Germany). The erythroid differentiation is induced with human recombinant erythropoietin (Epoietin beta, Neorecormon Roche, Grenzach-Whylen, Germany). The primary antibodies used were directed against: GATA-1, GATA-2, PU.1, α-, β-, and γ-globin (Santa Cruz Biotechnology), SphK1, PI3K, P-PI3K^Y458, AKT, P-AKT^S473, P-ULK1^S758, mTOR, P-mTOR^S2448, Atg7, Atg5-Atg12, Atg13, P-Atg13^S355, Beclin-1, p62/SQSTM1 (Cell Signaling, Leiden, The Netherlands), LC3 (Sigma), Rubicon (Abcam, Cambridge, UK), and ceramide MID 15B4 (Enzo Life Sciences, Antwerpen, Belgium), and the antibodies used for flow cytometry (CD235a/GPA, CD11b) were purchased from BD BioSciences (Erembodegem, Belgium).

Thapsigargin, phorbol 12,13-dibutyrate, C2-ceramide and C6-ceramide and C2-dihydroceramide were purchased from Sigma-Aldrich. FuGENE HD was from Promega (Madison, USA), and TransIT/Jurkat was from Mirus Corporation (Madison, USA). mAb OKT3 and mAb 4G10 were from Thermo Fisher Scientific and A488-conjugated goat anti-mouse γ2b was from Invitrogen. The genetically encoded Ca²⁺ indicator RGECO-1 (Zhao et al., 2011 (link)) was purchased from Addgene (plasmid #32444; Cambridge, USA). A488-CTxB was from Invitrogen and TX-100 Surfact-Amps was from Thermo Fisher Scientific.