Siramesine

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).

The immortalised mouse microglial cell line BV2 was obtained from the American Type Culture Collection and cultured with DMEM containing 10% FBS. Galectin‐3‐GFP plasmid was a kind gift from Prof. Tamotsu Yoshimori (Maejima et al, 2013). BV2 cells were plated in glass coverslips and transfected using Lipofectamine 2000 (Life Technologies) according to the manufacturer's instructions. 48 h after transfection, the cells were treated with SM (40 μM, Sigma‐Aldrich) or with the ASM inhibitor siramesine (30 μM, Sigma‐Aldrich) during 24 h. Images were taken on a confocal LSM510 microscope (Carl Zeiss).

The following antibodies were used: rabbit anti-P2X7 (Alomone Labs), mouse anti-flotillin-1 (BD Bioscience), and rabbit anti-Ki67 (Novocastra). Phalloidin-488 was used to stain F-actin; the monoclonal antibody against desmoyokin-AHNAK (dA) was a gift of Prof. Jacopo Meldolesi (Università Vita-Salute San Raffaele, Milano, Italy). Rabbit anti-Alix (Millipore), goat anti-CD63 (Biorbyt), mouse anti-COX-IV (Cell Signaling Technology), rabbit anti-cleaved caspase-3 (Cell Signaling Technology), β-actin (Sigma). Oxidized ATP (oxATP), brilliant blue G (BBG), probenecid, imipramine, siramesine and actinomycin D (actD) and ethidium bromide were purchased from Sigma-Aldrich; ARL67156 and SR11302 were from Tocris. ¹⁰Panx was from Innovagen and 11R-VIVIT from Calbiochem. WP631 was a gift of Dr. Cinthia Farina (San Raffaele Scientific Institute, Milano, Italy).