Hela tet on 3g cell line

The transfection and selection of the pTRE-LTR HeLa Tet-On^® 3G Cell Line (HeLa-iLTR) was performed following the manufacturer’s instructions (Clontech). For RNA decay assays, 1 × 10⁵ HeLa-iLTR cells were seeded into 6-well plates 24 h prior to transfection with 1 µg pCDNA3.1, pCMV-NF45 or pEF6-NF90 using GenJet™ transfection reagent (SignaGen Laboratories, Rockville, MD, USA) following the manufacturer’s instructions. At 24 h post transfection 10 ng/mL of doxycycline was added to induce expression of HIV RNA. After 12 h of induction, the cells were washed 3 times and returned to cDMEM. Total RNA was harvested at 0, 8, 16, 24 and 32 h (Omega Biotech, Norcross, GA, USA). RNA was reverse transcribed using random primers and cDNA was amplified using primers (5′-TCTGGCTAACTAGGGAACCCAC-3″ and 5′-CTGACTAAAAGGGTCTGAGG-3′). Primers designed to 18S RNA were used as an internal control (5′-GTAACCCGTTGAACCCCATT-3′ and 5′-CCATCCAATCGGTAGTAGCG-3′).

The HeLa Tet-On^® 3G Cell Line (Clontech; 631183), which expresses the tetracycline (Tet)-regulated transactivator Tet-On 3G, was established as a parental cell line. Cells were grown in DMEM with 10% FBS, and 250 μg/ml G418 (Gibco; 11811031) was added to maintain the transfected vector. The stable EWSR1-FLI1 HeLa clones grew in the same medium but supplemented with 0.5 μg/ml of puromycin (SIGMA; A1113803). Both antibiotics were removed from the medium culture prior to carrying out experiments. Doxycycline (Clontech; 631311) was used to induce the system. The ES cell lines A673 and RDES were obtained from ATCC (#CRL-1598 Lot 58078570 and #HTB-166 Lot 58078725, respectively). Cells were grown on 0.1% gelatin-coated plates in DMEM 10% FBS (A673) and RPMI 15% FBS (RDES). All cell lines were maintained in 37°C incubators, in an atmosphere of 5% CO₂. Cell lines were free of mycoplasma, and were continuously screened with the MycoAlert^® Mycoplasma Detection Kit (Lonza) to ensure this.

The HeLa Tet-On^® 3G cell line (Clontech, #631183) was used to achieve inducible and regulated expression of EWSR1-FLI1. This cell line constitutively expresses the reverse tetracycline-responsive transcriptional activator characterized by high sensitivity to doxycycline (Clontech, #631310). For this, the EWSR1-FLI1 (7–6 type) cDNA (in which the original stop codon was replaced for a triple FLAG-stop codon sequence) was first cloned into the response vector pRetroX-Tight-Pur (Clontech, #632104). After retroviral infection, cells were selected using 250 ゼg/ml G418 (Gibco, #11811–031) and 0.6 ゼg/ml puromycin (SIGMA, #18833), to obtain stably-transfected individual clones that conserved both plasmids. Finally, individual clones were selected that had no leakage and were capable of expressing ectopic EWSR1-FLI1 upon doxycycline induction. EWSR1-FLI1 expression was analyzed by Western blot using an antibody against the COOH-terminal region of FLI1, which was present in this chimeric protein (clone C-19) (Santa Cruz, #SC-356).