Lipofectamine rnaimax

Transient transfection assays were performed in 6-well plate (2.5 x 10⁵ cells/well) using Lipofectamine 2000 (Invitrogen, 11668–027) in Opti-MEM medium (Gibco, 51985–026) according to the manufacturer’s instructions using 1 μg of plasmid. Three hours post-transfection, Opti-MEM was replaced by complete DMEM and cells were kept overnight at 37°C and used for downstream infection assays.
For siRNA mediated depletion, U2OS cells were seeded in 6-well plate (2.5 x 10⁵ cells/well) and two consecutive rounds of siRNA transfection were done 24 h and 48 h post seeding. One hundred pmol of each siRNA were transfected using Lipofectamine RNAi max (Invitrogen, 13778–030) in Opti-MEM according to manufacturer’s instructions. Three hours post-transfection, Opti-MEM was replaced by complete DMEM and cells were transfected again the next day. The poly(I:C) treatment was performed 3 h by transfecting 2 μg/mL of poly(I:C) (Invivogen, tlrl-pic) using the Lipofectamine RNAi max protocol.

NSC34^{50 (link)}, COS-7^{51 (link)}, and HEK-293^{52 (link)} cells were maintained at 37 °C and 5% CO₂ in Dulbecco’s modified high glucose Eagle’s medium supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic (Thermo Fisher Scientific). For imaging, cells were plated on glass coverslips coated with poly-L-lysine (50 μg/ml) and bovine plasma fibronectin (10 μg/ml) (Life Technologies). Plasmid transfections were done using Lipofectamine 2000 (Invitrogen) in Opti-MEM media (Thermo Fisher Scientific). Cells were imaged or harvested 48 h post-transfection. V1-ER and V2-Mito reporter plasmids were transfected at a 1:1 ratio at a total DNA of concentration of 1 μg per well of a 12 well dish (except where otherwise indicated). MFN2 siRNA (Dharmacon) transfection was performed with lipofectamine RNAiMAX at an siRNA concentration of 50 nM for 48 h.
Stably transfected NSC34 cells co-expressing V1-ER and V2-Mito were selected on 400 μg/ml zeocin (InvivoGen).
ER stress was induced with tunicamycin (Calbiochem) at 2 μg/ml or 10 μg/ml for 6 h, with an equivalent volume of DMSO as vehicle only control.