Lentiviral pTRIPz vectors encoding Tet-inducible sh-HK2 (RHS4696) and nonsilencing control (RHS4743) were purchased from Open Biosystems (Thermo Fisher Scientific, Inc., Carlsbad, CA, USA). These plasmids contain RFP cassette downstream of the Tet/ON promoter.
To produce the infectious viruses, 293T packaging cell line was co-transfected using calcium phosphate with the following retroviral backbone plasmids: the TRIPz-RFP/sh-HK2 vectors (a pool of three siRNA sequences), the packaging plasmid pCMVΔ8.2 and the envelope plasmid pVSV-G.
After 48 h of incubation, the virus particles in the medium were collected and filtrated (0.45 μm filter). Neuroblastoma lung metastatic cells were plated (2 × 106) 24 h before infection. The cells were infected in the presence of 1 μg ml−1 Polybrene overnight and the virus-containing medium was replaced with fresh medium. After 72 h, 1 μg ml−1 Puromycin (InvivoGen, San Diego, CA, USA) was added for 7 days in order to stably select infected cell population. After selection, Puromycin was regularly added to the cultures.
The Tet-On mode of sh-HK2 infected cells was activated by the addition of 1 μg ml−1 Doxycycline (Sigma-Aldrich). Efficiency of infection was examined by visualising RFP in Leica DMRB Fluorescence Microscope (Leica Biosystems, Nussloch GmbH, Germany). Efficacy of sh-HK2 inhibition was verified by western blotting.
To produce the infectious viruses, 293T packaging cell line was co-transfected using calcium phosphate with the following retroviral backbone plasmids: the TRIPz-RFP/sh-HK2 vectors (a pool of three siRNA sequences), the packaging plasmid pCMVΔ8.2 and the envelope plasmid pVSV-G.
After 48 h of incubation, the virus particles in the medium were collected and filtrated (0.45 μm filter). Neuroblastoma lung metastatic cells were plated (2 × 106) 24 h before infection. The cells were infected in the presence of 1 μg ml−1 Polybrene overnight and the virus-containing medium was replaced with fresh medium. After 72 h, 1 μg ml−1 Puromycin (InvivoGen, San Diego, CA, USA) was added for 7 days in order to stably select infected cell population. After selection, Puromycin was regularly added to the cultures.
The Tet-On mode of sh-HK2 infected cells was activated by the addition of 1 μg ml−1 Doxycycline (Sigma-Aldrich). Efficiency of infection was examined by visualising RFP in Leica DMRB Fluorescence Microscope (Leica Biosystems, Nussloch GmbH, Germany). Efficacy of sh-HK2 inhibition was verified by western blotting.