Lentiviral lkb1 short hairpin rna shrna constructs

For generation of stable knockdown cells, lentiviral LKB1 short-hairpin RNA (shRNA) constructs and a non-targeting shRNA control were purchased from Open Biosystems (Huntsville, AL). These constructs were cloned into a pLKO.1 vector. HEK293T cells (2 × 10⁶ cells) were plated on 100 mm dishes and one day later were co-transfected with 5 μg shRNA constructs, 3 μg pCMV-dR8.2 dvpr (Addgene) and 2 μg pCMV-VSV-G (Addgene) using a calcium phosphate transfection kit (Invitrogen). After 6 h, the medium was exchanged with fresh medium, and then the viral stock was collected for 1–2 days and filtered to remove non-adherent HEK293T cells. To obtain stable shRNA expressing clones, Calu-6 or H358 cells were plated on six-well plates, and one day later infected with 1 ml of lentiviral suspension plus 4 μg/ml polybrene. Puromycin selection (0.5–2 μg/ml) was initiated 2 days after lentiviral infection.