P ulk1s758

Cucurbitacin E was obtained from Shanghai Shunbo Chempharm Co (Shanghai, China). Chloroquine (CQ), dimethylsulfoxide (DMSO) and sodium dodecyl sulfate (SDS), polyformaldehyde were from Sigma-Aldrich (St. Louis, MO, USA). WST-1 was from Roche (Penzberg, Germany). Lipofectamine RNAiMAX, Dulbecco's modified Eagle's medium (DMEM), penicillin, streptomycin, L-glutamine, and fetal bovine serum (FBS) were from Gibco/ Invitrogen (Carlsbad, CA, USA). Polyvinylidene difluoride (PVDF) membranes (Hybond-P) were obtained from GE Healthcare Life Sciences (Piscataway, NJ, USA). Antibodies against p-p70S6K_T389, p70S6K, p-4E-BP1_T37/46, 4E-BP1, p-S6_S235/236, S6, p62/SQSTM1, LC3B, ATG5, Beclin 1, p-AKT_S473, p-Akt_T308, Akt, p-AMPKα_T172, AMPKα, p-AMPKβ1_S108, AMPKβ1, p-ULK1_S758, p-ULK1_S555, ULK1, p-Raptor_S792, Raptor, β-tubulin and HRP-conjugated second antibody were obtained from Cell Signaling Technology (Danvers, MA, USA). The antibody against LAMP2 was obtained from Abcam (Cambridge, MA, USA).

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).