Rhs4743

To generate the MDA‐MB‐231, HMLE and MCF10A TRIPZ shPRP4K cell lines TRIPZ (shPRP4K‐1 = clone: V2THS_383962, shPRP4K‐2 = clone: V3THS_383960, Non‐silencing shCTRL = RHS4743), lentiviral shRNAs were purchased from ThermoFisher Scientific (Thermo; Mississauga, ON, CA). Lentivirus was produced by co‐transfection of the TRIPZ shRNA with pMD2.G and psPAX2 (gifts from Didier Trono, Addgene plasmid #12259 and #12260, respectively) lentiviral packaging vectors into human HEK‐293T cells via calcium–phosphate transfection (Promega; Madison, WI, USA), according to the manufacturer's directions. After 48 h, media from the transfected cells were filtered using a 0.45 µ filter, and the viral media added to the target cell line for 48 h. Following the 48 h, the cells were split and treated with virus for another 48 h to increase the transfection efficiency. Cells were allowed to recover in fresh media for 24 h followed by selection in fresh medium containing 2 µg/ml puromycin for 4–5 days. To induce expression of the inducible PRP4K shRNA, 5 µg/ml doxycycline (Sigma‐Aldrich) was added to culture media for 96 h prior to experimentation with the drug being replaced every 24 h.

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.

Two different TET1 shRNAs in the pTRIPZ vectors (shTET1#1: V2THS_141063 and shTET1#2: V2THS_203196, Open Biosystems) were transferred into MulI and XhoI sites of the pGIPZ vectors (Open Biosystems). A non-targeting shRNAmir-pGIPZ vector was used as a negative control (RHS4743, Open Biosystems). To produce lentiviral particles, pGIPZ-shTET1 and package plasmids psPAX2 and pMD2.G (Addgene) were transfected into HEK293FT cells (Invitrogen) at a ratio of 1:1:1 using Lipofectamine^®2000 Transfection Reagents (Invitrogen). Two days after transfection, the viral supernatant was collected and filtered with 0.45 μm filters (Millipore). HEK293T cells were then infected with each lentivirus supernatant in the presence of 8 μg/ml of polybrene (Sigma). Puromycin selection (1.5 μg/ml, Sigma) began 2 days after infection. Stable knockdown cells were cloned by limiting dilution and selected by western blot assay based on TET1 protein level. These knockdown cells were subjected to further analyses 3–4 months after transfection.