C2 ceramide

Human BeWo choriocarcinoma cells (which have a villi syncytiotrophoblastic phenotype) were maintained in F12K media supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin. Cells were plated at a density of two hundred thousand cells per well cm in six-well plates. Cells were treated with C2-ceramide (1 μM; Sigma-Aldrich, St. Louis, MO), insulin (50 nM, Sigma-Aldrich, St. Louis, MO) or fresh media for 24 h. Importantly, C2-ceramide is an oft-used agent, due to its solubility. After treatment, BeWo cells were used for mitochondrial respiration determination. Cell lysates were collected and evaluated for active caspase 3 and XIAP immunoblot determination.

The role of ERK signalling in reversal of senescence was investigated using agonists (ceramide) and inhibitors (trametinib) of the ERK pathway. Cells from a senescent culture were seeded at 6 × 10⁴ cells/cm² in a 6 well plate in serum free media, and after 10 days were treated with 1-20 μM of the ERK inhibitor trametinib (LC laboratories, Woburn, USA for 24 h hours, or with the ERK agonist N-Acetyl-D-sphingosine (C2-ceramide; Sigma Aldrich, UK) at 20 μM for 24 or 120 h. To examine the role of ERK signalling in resveralogue-induced rescue of senescence, HNDF cells were treated with 20 μM of the ERK agonist C2-ceramide as above, but with the addition of 5 μM resveralogue for 24 h.

tdTomato and Lifeact-GFP were purchased from ibidi, Inc. Ser3 peptides with the sequence of MAS(p)GVAVSDGVIKVFN were synthesized by GenScript (GenScript). GW4869, desipramine, fumonisin B1, C2-ceramide, and Pyrazolopyrimidine 2 (PP2) were purchased from Sigma-Aldrich (Sigma-Aldrich, USA). Recombinant IL-1β (PeproTech) was dissolved in DMEM and used after one freeze–thaw cycle at 50 ng/ml. After 14–18 days in vitro (DIV), neurons were treated at 37 °C with 50 ng/ml IL-1β, with control neurons receiving equal volumes of vehicle. cLTP was induced as described previously [24 (link), 25 (link)]. Briefly, hippocampal neurons were maintained in normal ACSF (5 mM HEPES [pH 7.3], 125 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, and 33 mM glucose). Osmolarity was adjusted to 290 mosmol/l. Chemical LTP was induced by changing the medium to Mg²⁺-free ACSF (5 mM HEPES [pH 7.3], 125 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 33 mM glucose, 0.2 mM glycine, 0.02 mM bicuculline, and 0.003 mM strychnine) for 10 min. After that, the incubation solution was altered back to control solution without glycine for 20 min before surface GluA1 labeling and for 30 min before fixation for immunohistochemistry to detect changes in F-actin, respectively. Neurons were treated with vehicle or IL-1β before (1 h), during (10 min), and after cLTP stimulation at indicated time points.