Transfection reagent

Replication incompetent lentivirus were produced in HEK 293T cells co-transfected by mixing 5 μg of either pLKO.1 Empty Vector or pLKO.1 shRNA targeting SMARCD3 with 6 μg Lenti-vpak packaging kit components (OriGene) and 33 μL of transfection reagent (OriGene) in 1.5 mL OptiMEM, incubated for 20 min at RT. This transfection mixture was added onto 10 mL of HEK 293T cells at 2×10⁵ cell/mL in fresh culture medium was transferred. Culture medium was replaced 24h post-transfection and viral supernatants were harvested twice at 48 and 72h and combined. Viral supernatants were clarified by centrifugation at 500 g, filtered through a 0.45 μm PES and stored at −80°C. Lentiviral transduction was performed with 5×10⁵ cells seeded into a T25 flask in media supplemented with μg/mL polybrene. Viral supernatant (500 μL) was added onto the cells and they were then incubated for 24h. 72h post-transduction, cells were selected with Puromycin (for pLKO) at 1 μg/mL. After two weeks of Puromycin treatment cells were considered selected. Knockdown efficiency of SMARCD3 was determined by Western Blotting and/or qRT-PCR as described below. Cells transduced with FUCCI plasmids were sorted by FACS to select homogenous positive cells populations.

Two different formats of Sirt1 siRNA were applied 48 hours before ICH, in order to enhance the silencing effect. An intracerebroventricular injection (I.C.V) was then performed as previously described^{14 (link)}. The Sirt1 siRNA or scramble siRNA mixed with the transfection reagent (OriGene Technologies) (100 pmol/2 µl) was delivered into the ipsilateral ventricle with a Hamilton syringe and administered over 2 minutes. After the needle was removed, the burr hole was sealed with bone wax. The incision was closed with sutures and the mice were allowed to recover.