Staurosporine

Jurkat cells were cultured in RPMI 1640 medium without FCS for 16 h, and then treated with staurosporine (0.5 μg/ml, SIGMA), followed by an incubation at 37 °C for 3 h. staurosporine treatment yielded a population of 83% annexin V + cells. Apoptotic cells (AC) were resuspended at a concentration of 1 × 10⁶ cells/ml and labeled with CellTrace™ Violet reagent (0.5 μM, Invitrogen, Thermo Scientific) for 20 min. For efferocytosis, macrophages were cultured with labeled AC (ratio 1:4) in p24 plates during 1 h at 37 °C. After 1 h, macrophages were rinsed with PBS to remove unbound AC and detached with PBS 5 mM EDTA, pelleted by centrifugation and fixed (IOTest 3 Fixative Solution, Beckman Coulter) before analyzing by flow cytometry.

Upon acquisition of baseline, neurons were either treated with 1.5 µM staurosporine (Sigma-Aldrich) to induce apoptosis or kept under control conditions. In a sub-set of experiments, neurons were additionally treated with caspase inhibitor (100 µM, Z-DEVD-FMK, R&D Systems), and the myosin blocker BDM (2,3-Butanedione menoxime, Sigma-Aldrich, 20 mM) in the presence of staurosporine.
For experiments in which confocal imaging preceded SMLM acquisition, H2B::mCherry-transduced primary cortical neurons were either imaged as control (1.6 µM NucView and imaging for 1 h) or treated with staurosporine. In this case, 1.0–1.5 µM staurosporine was applied to the dishes 0 to 4 h before the acquisition started, then 1.6 µM NucView was added and the neurons were imaged for up to 6 h. All cells were directly fixed with 4% PFA afterwards.

After 4 days of spheroid growth, 20 μl culture medium containing 80 nl compounds (ENZO Screen-Well ICCB Known Bioactives library, Enzo Life Sciences, Farmingdale, NY, USA (468 compounds), final compound dilution of 0.1–20 μM, depending on original stock concentration) were added and incubated for additional 3 days either at normal culture conditions (21% O₂, 37 °C, 5% CO₂) or in a hypoxic chamber (<1% O₂, 37 °C, 5% CO₂). A 0.2% DMSO solution was used as solvent control and Staurosporine (Sigma-Aldrich) as general toxic control (10 μM).
Screening hits and further tool compounds, including Staurosporine, Antimycin A, 2-DG, Fluphenazine, Chlorpromazine, Thioridazine, Clozapine, Bafilomycin A, Siramesine, N-Palmitoyl-D-sphingomyelin, SM (from bovine brain), Ceramide (from bovine spinal cord) and 2-Dioleoyl-sn-glycero-3-phosphocholine (18:1 (Δ9-Cis) PC), were purchased from Sigma-Aldrich. 1-Stearoyl-2-arachidonoyl-sn-glycero-3-phosphocolin (18:0–20:4 PC) and 1-stearoyl-2-docosahexaenoyl-sn-glycerol-3-phosphocolin (18:0–22:6 PC) were purchased from Avanti Lipids (Alabaster, AL, USA). All compounds, except for SM and PCs, were dissolved in DMSO (10 mM) and stored at −20 °C. SM and PCs were dissolved in ethanol. Hypoxia mimicking agent DFO (Sigma-Aldrich) was used at a final concentration of 1 mM (2D growth assays for 16–24 h).

Rat hippocampal or cortical neurons were treated with 1 µM dexamethasone (Sigma) for 24 h. The cells were then harvested, extracted and analyzed for expression of CPE-ΔN. FGF2 (R&D) or BDNF (Promega) was added to neurons for 15 min. The cells were then harvested, extracted and analyzed for activation of ERK and AKT. In other experiments, primary neurons were transduced with adenoviral vectors (Type 5 (dE1/E3)), carrying the cDNA of CPE ΔN (custom made by Vector Biolabs) or LacZ (Vector Biolabs) as a negative control, at 50 MOI for 72 h. The neurons were then treated with 100 µM H₂O₂ (Sigma), 40 µM glutamate (Sigma) or 0.4 µM staurosporine (Sigma) for 24 h. In specific experiments, 10 µM LY294002 (Cell Signaling), 5 µM U0126 (Cell Signaling), 1 µM PD166285 (Sigma) or SU5402 (Sigma) were added to the neurons during the 20–24 h treatment with glutamate, staurosporine or H₂O₂. Cell viability, cell cytotoxicity and TUNEL assays were then used to measure cell death after the treatments.

Antibodies used were Mouse Anti-HA (Sigma, H9658), AP-3 (SA4, Developmental Studies Hybridoma Bank) and APP C-terminal (Sigma, A8717). The PKC activator Phorbol-12-myristate-13-acetate (PMA) was purchased from Sigma (P8139) and 8-[2-(2-pentyl-cyclopropylmethyl)-cyclopropyl]-octanoic acid (DCP-LA) was purchased from (Sigma, D5318). Staurosporine was purchased from Millipore (Cat No. 569397). Gö6976 was purchased from Tocris Bioscience (Cat. No. 2253).