On targetplus human app 351 sirna

Cryopreserved primary human hepatocytes (PHH) (BioIVT) and primary human stellate cells (HSCs; Lonza) were co‐cultured in ultra‐low attachment plates (Sigma) at a ratio 4:1 as previously described.^[73 (link)
^] Spheroids were treated for 1 week with a mixture of oleic and palmitic acid (400 µm of each). For APP knock‐down experiments, cells were transfected with ON‐TARGETplus Human APP (351) siRNA (Dharmacon) at a final concentration of 50 nm. Thereafter the spheroids were processed for qPCR and immunofluorescence analyses as described in Supporting Information.

Transient knockdown experiments were performed following the Reverse-Transfection of adherence cells protocol adapted from Qiagen (Qiagen, Hilden, Germany). Briefly, siRNAs and Lipofectamine^® 2000 (Invitrogen, Carlsbad, CA, USA) were diluted in Opti-MEM (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and incubated for 20 min at room temperature to allow the formation of the transfection complex. Confluent cells were then harvested and seeded on top of silencing complexes in 60 mm (3.5 × 10⁵ cells) or 100 mm (6.5 × 10⁵ cells) or 6-well plate dishs (1.8 × 10⁵ cells). Cells were transfected for 48 h under their normal growth conditions (EGM-2, 10% FBS) with 20 nM siRNA control (Negative Control siRNA, Qiagen #0001027310), or 20 nM siAPP-A (Hs_APP_8 FlexiTube^® siRNA, Qiagen #SI02776893), or 10 nM siAPP-B (Hs_APP_10 FlexiTube^® siRNA, Qiagen #SI02780288), or 20 nM siAPP-C (ON-TARGETplus Human APP (351) siRNA, Dharmacon #J-003731-06-0002). All results showed in main figures refer to HUVEC cells transfected with 20 nM of siAPP-C (Dharmacon Inc., Lafayette, CO, USA).