Lentiviral shrna constructs

The lentiviral shRNA constructs were purchased from Thermo Scientific (Pittsburgh, PA, USA). Lentiviruses were produced by co-transfection of shRNA-expressing plasmid, envelope plasmid (pMD.G) and packaging plasmid (pCMV-dR8.91) in 293T cells using calcium phosphate (Invitrogen, Carlsbad, CA, USA). The 293T cells were incubated for 18 h, and the culture medium was then removed, and refreshed. The viral supernatants were harvested and tittered at 48 and 72 h post-transfection. Monolayer cells were infected with the lentiviruses in the presence of polybrene, and were further selected using puromycin.

Lentivirus-based production and infection followed a previously published protocol [30 (link)]. Briefly, the lentiviral shRNA constructs were purchased from Thermo Fisher Scientific (Pittsburgh, PA, USA). Lentiviruses were produced by calcium phosphate transfection method (Invitrogen) and created by co-transfection of shRNA-expressing plasmids, envelope plasmids (pMD.G) and packaging plasmids (pCMV-dR8.91) in 293T cells. The transfected 293T cells were incubated in the culture medium for 18 h; the supernatant was then removed and refreshed. The supernatants containing the shRNA viruses were harvested and tittered at 72 h post-transfection. CRC cells were infected with the lentiviruses in the presence of Polybrene (8 μg/mL), and were proceeded enrich infected cell by puromycin selection (2 μg/mL).

To knock down endogenous hLIF, lentiviral shRNA constructs were obtained from Open Biosystems (Thermo Scientific, Waltham, MA, USA). 293T cells (1.5 × 10⁶) were subcultured in a 10 cm dish. Twenty-four hours after cell seeding, the lentiviral vector encoding hLIF shRNA (3 μg), packaging vector pCMV-VSVG (0.3 μg), and the envelop plasmid pHRCMV-8.2 ΔR (3 μg) were transfected in 293T cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). Forty-eight hours after transfection, the supernatant harboring lentiviruses was collected and filtered using a sterilized 0.45 μm syringe filter. LIF shRNA lentiviral supernatant with polybrene (5 μg/mL) was added to the Ishikawa cells. To select cells with stable integration of LIF shRNA, the viral infected Ishikawa cells were treated with 3 μg/mL puromycin for 1 week. The knockdown efficiency of the LIF shRNA was verified by western blot analysis. The best hLIF shRNA #5, among 5 hLIF shRNA clones (#3–#7), was used for subsequent experiments. The sequence of LIF shRNA #5 used for knocking down LIF was 5’-TTACCCGAGGTGTCAGGGCCG-3’.