C2 ceramide

SH-SY5Y cells were treated with cell-permeable, biologically active ceramide, C2-ceramide, at a 10 to 50 μM concentration for various times up to 24 h. In most of the experiments, the cells were treated with the following compounds: PJ-34 (20 μM), PARP-1 inhibitor; α-pifithrin (20 μM), p53 inhibitor; cyclosporine A (2 μM), inhibitor of the permeability transition pore; UO126 (1 μM), ERK1/2 kinase inhibitor; SP600125 (5 μM), JNK kinase inhibitor; sphingosine-1-phosphate (1 μM), LY294002 (50 μM), PI3-K inhibitor and Z-DEVD-FMK (100 μM), caspase-3 inhibitor added 1 h before incubation with C2-ceramide (25 μM). Our data showed that most of these compounds had no effect on cell viability in the control condition, with the exception of PARP-1 inhibitor, JNK kinase inhibitor and PI3-K inhibitor, which significantly decreased SH-SY5Y cell survival (by about 20 %, PJ-34, SP600125; 35 %, LY294002). The above-mentioned compounds were purchased from the following: C2-ceramide and sphingosine-1-phosphate from Enzo Life Sciences, LY294002 from Cell Signalling Technology, PJ-34, α-pifithrin, SP600125, U0126 and cyclosporine A from Sigma-Aldrich, Z-DEVD-FMK from Tocris Bioscence.

Rabbit polyclonal anti-myc antibody, mouse monoclonal anti-actinin-1 antibody, and DMSO were from Sigma–Aldrich (St Louis, MO, USA). Mouse monoclonal anti-Stx6 antibody was from BD Transduction Laboratories (San Jose, CA, USA). Rabbit polyclonal anti-Stx6 and anti-Stx16 antibodies were from Synaptic Systems (Goettingen, Germany). Mouse monoclonal anti-Tubulin antibody was from Abcam (Cambridge, MA, USA). Human holo-transferrin conjugated to A488 was from Invitrogen (Grand Island, NY, USA). Mouse anti-myc (c-Myc 9E10) and rabbit anti-furin (H-220) were from Santa Cruz Biotechnology (Dallas, TX, USA). Polyclonal anti-P-Akt(308) and P-Akt(473) were obtained from Cell Signaling Technology (Danvers, MA, USA). Cy3- and A488-conjugated donkey anti-rabbit and donkey anti-mouse secondary antibodies and horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibodies were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Nocodazole was purchased from EMD Biosciences Inc. (Darmstadt, Germany) (10 mM stock in DMSO) and C2-ceramide was purchased from Enzo Life Sciences (Farmingdale, NY, USA) (50 mM stock in DMSO). Pre-designed siRNA for Stx6 (siStx6: 5′-CCGAGTCATCAGAAGAACTAA-3′) and non-related (siNR: 5′-AATAAGGCTATGAAGAGATA C-3′) were from Qiagen (Valencia, CA, USA). Human insulin was purchased from Eli Lilly (Indianapolis, IN, USA).