Hek293t packaging cells

To knockdown APC expression and construct a stable cell line, pCDH-CMV-MCS-EF1-Puro (CD510B-1) shRNA vectors targeting human APC was purchased from Open Biosystems (Thermo Fisher Scientific, US). The sequences were based on the previous siRNA study. Lentiviral shRNA was produced by co-transfection of the trans- lentiviral packaging system by adding mixture of pCDH-CMV-MCS-EF1-Puro (CD510B-1)-shAPC and packaging plasmids pSPAX2 and pMD2.0G into HEK293T packaging cells (Open Biosystems, US). Viral supernatants were collected for cell infection, supplemented with 6 μg/mL polybrene. After 24 hours incubation, HCT116 and NCM460 were transduced by the lentiviral particles followed by puromycin selection (1.5μg/mL) for 7 days. The cells stably expressing shRNA were maintained in puromycin (0.75 μg/mL).

To knockdown STMN-1 expression, pGIPZ-lentiviral shRNAmir vectors targeting human STMN1 and Non-silencing pGIPZ control vector were purchased from Open Biosystems (Thermo Fisher Scientific, Inc.). The sequences of STMN-1 shRNA are as following: 5′-TTATTAGCTTCCATTTTGT-3′; 5′-TCTCTTCTATTGCCTTCTG-3′ and 5′-TTATTAACCATTCAAGTCC-3′. Lentiviral shRNA was produced by Co-transfection of the Trans-Lentiviral packaging mix with a shRNA transfer vector into HEK293T packaging cells (OpenBiosystems). For cell infection, viral supernatants were supplemented with 6 μg/mL polybrene and incubated with cells for 24 hours. Esophageal adenocarcinoma cell lines were transduced by the lentiviral particles followed by puromycin selection (1 μg/mL) for 10 days. The cells stably expressing shRNA were maintained in puromycin (0.2 μg/mL).

pGIPZ-lentiviral shRNAmir vectors targeting human Nox4 gene and nonsilencing pGIPZ control vector were purchased from Open Biosystems (Thermo Fisher Scientific, Inc.). pGIPZ cloning vector contains Turbo GFP reporter and expresses a puromycin-resistant gene. Lentiviral shRNA was produced by cotransfection of the Trans-Lentiviral Packaging Mix with a shRNA transfer vector into HEK 293T packaging cells (Open Biosystems). Supernatants containing either the lentivirus expressing the Nox4 shRNA or the control shRNA were harvested 72 h after transfection. The lentiviruses were purified using ultracentrifugation, and the titer of the lentiviruses was determined. U87MG and U251 cells were transduced by the lentiviral particles at a multiplicity of infection (MOI) of 10 followed by puromycin selection for 10 days. The clones stably transfected with pGIPZ-lentiviral shRNAmir was referred to as Nox4 shRNA cells, whereas the cells stably transfected with pGIPZ nonsilencing control vector as scrambled cells. The knockdown of Nox4 was evaluated by real-time quantitative PCR and Western blot analysis.