Optimem

Plasmids were purified using Endotoxin-Free Maxiprep Kit (Qiagen). HEK293T cells were seeded at 3.8 × 106 cells per plate in DMEM complete in 10 cm tissue culture plates and incubated at 37 °C, 5% CO₂ overnight. The next morning, media was replaced with fresh complete DMEM containing 25 µM chloroquine diphosphate and incubated for 5 h. 27 µg of total plasmid DNA as a mixture of the transfection plasmids RRE:VSVG:REV:pHIV in ratio of 2:1:2:4 was diluted in 500 µl of OptiMEM (Gibco) and mixed with jetPEI (Polyplus)-OptiMEM (µg DNA:µg PEI − 1:3). After 20 min incubation transfection mix was added to 293 T packaging cells, incubated overnight and media replaced the next morning. Virus was harvested at 48 and 72 h, centrifuged at 1000 × g for 5 min and filtered through a 0.45 µm PES filter, pooled and added to HEK-293T cells. The media was replaced after 48 h and cells were cultured prior to reaching confluency. Stable expression of recombinant proteins was confirmed by fluorescent microscopy and immunoblotting.

Cells were seeded in 6-well dishes at 70% confluence and transfected with siRNA against ATL2 (Bioneer, Daejeon, South Korea) using OptiMEM and INTERFERin (Polyplus-transfection), according to the manufacturer's instructions. Transfected cells were maintained for 24 h prior to further experiment. The sequences of siRNA are as follow:

ATL2 sense, 5'-UAG AGU UUG UUA CAG ACU G-3';

ATL2 antisense, 5'-CAG UCU GUA ACA AAC UCU A-3'.