Lentiviruses were produced as previously described with the following changes (99 (link)). HEK 293T cells were transfected with plasmids encoding psPax2, VSV-G, and various shRNAs using ProFection Calcium Phosphate Kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. One day after transfection, media was changed to low-serum media (5%) and incubated at 37°C for another two days. Cell culture media supernatant was collected and centrifuged at 24,000 rpm for two hours. The viral pellet was resuspended in 200 μL of DMEM. To infect cells, 50 μL of virus was added to the cells.
shRNAs and the NT9 control in a pLKO.1-puro vector backbone were purchased from Sigma-Alrich. The sequences of hairpin shRNAs were as follows: for Lat3 shRNA-1, 5′-CCGGCCCTGGAATCAAGCTGATCTACTCGAGTAGATCAGCTTGATTCCAGGGTTTTTG-3′; for Lat3 shRNA-2, 5′-CCGGGCTTCGGGTCATCTTCTATATCTCGAGATATAGAAGATGACCCGAAGCTTTTTG-3′.
shRNAs and the NT9 control in a pLKO.1-puro vector backbone were purchased from Sigma-Alrich. The sequences of hairpin shRNAs were as follows: for Lat3 shRNA-1, 5′-CCGGCCCTGGAATCAAGCTGATCTACTCGAGTAGATCAGCTTGATTCCAGGGTTTTTG-3′; for Lat3 shRNA-2, 5′-CCGGGCTTCGGGTCATCTTCTATATCTCGAGATATAGAAGATGACCCGAAGCTTTTTG-3′.