Virus production

For lentiviral production, HEK cells were transfected with the plasmids expressing the shRNAs together with the lentiviral packaging plasmid psPAX2(RRID:Addgene_12260) and the envelope plasmid pMD2.G (RRID:Addgene_12259) using TransIT-X2 (Mirus).

HEK 293 T cells were seeded in a 10-cm dish to reach 80% confluency the day after

transfection.The next day, the medium was changed.

On days 4 and 5 after transfection, the medium was collected and filtered through a0.45-

μm PVDF membrane.

To concentrate the virus, the filtered supernatant was centrifuged in a Vivaspin column (Sartorius Stedim Biotech) at 3000 RCF at 4 °C until the volume reached 500

–1000μl.

The virus-containing supernatant was collected, and the concentration

of p24 particles was determined with Lenti-X GoStix Plus (Takara).

Cells were infected with equal concentrations of p24 particles for the

scramble and respective shRNA.