Smase from staphylococcus aureus

Bodipy-sphingomyelin (Bdp-SM), Bodipy-ceramide (Bdp-cer), and Amplex Red kit were obtained from Molecular Probes (Eugene, OR). The purity of the above lipids was verified by thin-layer chromatography (TLC) on silicic acid-coated plates (Merck, Darmstadt, Germany) developed with chloroform/methanol/water (65 : 25 : 4, by vol.). The concentrations of Bdp-SM and Bdp-cer in chloroform were determined spectrophotometrically using 77,000 cm⁻¹ and 91,000 cm⁻¹, respectively, for their molar extinction coefficients. [³⁵S]-cysteine/methionine was obtained from Amersham Biosciences. Proanalysis grade solvents were from Merck, SMase from Staphylococcus aureus from Sigma, and other chemicals from standard sources.

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).

HeLa cells were obtained from American Type Culture Collection (ATCC) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37 °C in 5% CO₂ and 95% air incubator. Cells grown on glass coverslips were pretreated with 20 mM MβCD (Cyclolab), 1.7 unit/ml SMase from Staphylococcus aureus (Sigma), or DMEM/F12 medium without fetal calf serum as control at 37 °C for 30 min. The cells were then incubated with 5 μg/ml of bryoporin at 22 °C for 30 min and fixed with 4% paraformaldehyde in PBS. After blocking with 0.2% gelatine in PBS for 30 min, bryoporin was visualized with anti-His₅ antibody (QIAGEN) and Alexa488-conjugated antimouse IgG (Thermo Fisher Scientific). The specimens were observed under an LSM510 confocal microscope (Carl Zeiss) equipped with a C-Apochromat 63XW Korr (1.2 NA) objective. The effect of MβCD and SMase was confirmed by elimination of cell staining with filipin and Eqt-II-EGFP, respectively.