Tetracycline

For time course analyses, Tet-Off Advanced HeLa cells (Clontech) were grown in DMEM (Thermo Fisher) with tetracycline-free FBS (10%; Clontech), penicillin-streptomycin (1×; Thermo Fisher) and G418 (100 μg ml⁻¹; Corning). Before transfection, G418 was removed. A 6:1:1 mix of RBP–MCP, firefly–MS2 (or Renilla–MS2) and Renilla (or firefly) luciferase reporter (transfection control) constructs was diluted in 150 mM NaCl and mixed for transfection with polyethyleneimine (PEI; Polysciences) at a ratio of 1 μg DNA to 4 μg PEI. Cells were transfected at 50-60% cellular confluency, with a total of 125 ng and 250 ng DNA for 48-well plates and 24-well plates, respectively, and grown in the absence of G418. Reporter transcription was suppressed by the addition of tetracycline (1 μg ml⁻¹; Sigma) 48 h after transfection. Cells were lysed after 20, 80 and 120 min, and luciferase activity was measured with the Dual-Luciferase Reporter Assay System (Promega), following the manufacturer’s directions, in a microplate reader. Values were expressed as the ratio of the mean luciferase activity of MS2-tagged over MS2-untagged reporters from three replicates. For the screen and validations, transfections were done as for the time course assay, and luciferase activities were measured 48 h after transfection. Supplementary Table 2 lists the results of the luciferase assays.

Neuro 2a, Vero and Huh7 cells (acquired from American Type Culture Collection (ATCC)) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 200mM L-glutamine (L-glut; Sigma, 10,000 units/mL penicillin (Sigma), and 10 mg/mL streptomycin (Sigma). Tetracycline-inducible murine embryonic fibroblasts (MEF; Clontech) were maintained in the above medium supplemented additionally with 50 mg/mL G418. The generation of, and target gene induction from, Tetracycline-inducible murine embryonic fibroblasts overexpressing GFP, IFIT1 and Interferon-stimulated gene 20 (ISG20) has been previously described (38 (link)). BHK-21 cells (ATCC) were maintained in RPMI supplemented with 10% donor bovine serum (DBS), 10% tryptose phosphate broth (TPB), and supplements as above. All cells were grown at 37°C with 5% CO₂.

Silencing or overexpression of genes were performed as described previously (17 (link)). siRNAs were transfected at a concentration of 5nM to 20 nM using RNAi max (Invitrogen) (Supplementary Table-1). For ectopic expression, 500 ng of plasmid pCellFree_G03 empty vector or pCellFree_G03-FOXM1 were transfected in cancer cells or in normal epithelial cells. Stable knockdown of genes was performed using small hairpin (sh) RNA as described previously (14 (link)). Control and target specific shRNAs were purchased from Sigma to prepare lentiviral preparations (Supplementary Table-2). Viral particles were created by transfecting packaging vectors pLP1, pLP2 and VSVG plasmids including control empty vector pLKO.1 (Cat#SHC001V) or shRNA targets EGFR or ERBB2 purchased from Sigma-Aldrich (Saint Louis, MO) in HEK293T cells. Competent lentiviral particles were collected 48 h after transfection and used to infect target cells. To create a tetracycline inducible ZEB1 expressing cells, we cloned ZEB1 in pTRE-Tight GFP tetracycline inducible plasmid. GFP positive cells were selected after tetracycline (Takara Bio Inc., San Francisco, CA, USA) treatment.

To establish the stable overexpression of FXR1 in OVCAR3 cells, we transfected the cells with control vector or pReceiver-M39 vector expressing FXR1 (GeneCopoeia, Rockville, MD) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA). Cells were selected 4h after transfection to ensure that the cells were stably incorporated with control sequences or FXR1 using puromycin (2.5 μg/ml) containing culture media for two weeks. Western blotting was performed to check the expression of FXR1 and other targets in stable cells followed by colony formation assay for 12 days.
To create the tetracycline inducible cell line, HeyA8 cells were plated in a 6-well dish and transfected with pTRE-Tight GFP FXR1-expressing plasmid constructed at Vector Core facility, Versity Blood Research Institute using lentivirus method as mentioned above. GFP positive cells were selected by FACS sorting after tetracycline (Takara Bio Inc., San Francisco, CA, USA) treatment (1μg/ml). Transduced cells were expanded and used for further experiments.