On targetplus smartpool sirna against mouse

Microglia BV2 (gift from G. Brown, University of Cambridge), glioma C6 (ATCC® CCL-107™) and glioma GL261 (DSMZ ACC 802, gift from R. Glass, University hospital of Munich) rodent cell lines have been used in this study. Cells were regularly tested with Venor™GeM mycoplasma detection kit (Minerva Biolabs) and maintained in DMEM medium supplemented with penicillin (100 μg/ml), streptomycin (100 μg/ml) and 10% fetal calf serum (FCS) (all from Gibco™ ThermoFisher Scientific). Segregated cocultures were performed using a transwell system using the above mentioned medium with FCS reduced to 5%. Transfection of BV2 cells was carried out with Lipofectamine® 3000 (Invitrogen, ThermoFisher Scientific) following the manufacturer's recommendation. Predesigned ON-TARGETplus SMARTpool siRNA against mouse SIRT1 (L-048962-09) and mouse MOF (L-049440-05) as well as non-targeting control (D-001810) were purchased from Dharmacon. BV2 cells were treated with 50 µM EX527 (Tocris Bioscience) for 4 h, 5 µM SRT1720 (Santa Cruz Biotechnology) for 17 hours, or 250 nM MG132 (Sigma Aldrich) for 4 hours.