Puromycin

Cell culture media and supplements such as RPMI 1640, DMEM, PBS, and antibiotics (penicillin, streptomycin, and puromycin) were purchased from Lonza (Allendale, NJ), Fisher Scientific (Pittsburgh, PA) or Santa Cruz Biotechnology (Dallas, TX). Heat-inactivated fetal bovine serum (FBS) was obtained from Atlanta Biologicals (Flowery Branch, GA). Sodium butyrate and sanguinarine were procured from Sigma-Aldrich (St. Louis, MO). Trichostatin A (TSA), apicidin, HC toxin, LY294002, PX866, CAL-101, MK2206, Triciribine, GDC0068, Rapamycin, AZD8055, and BEZ235 were obtained from Cayman Chemical (Ann Arbor, MI). Tetrandrine was acquired from Santa Cruz Biotechnology, and (–)-depudecin was purchased from BioVision (Milpitas, CA) and MyBioSource (San Diego, CA). Datiscetin was ordered from BOC Sciences (Shirley, NY), while wortmannin and CUDC-907 were procured from Selleck Chemicals (Houston, TX).

To generate the ETS1-pLOC construct, a synthetic human ETS1 cDNA (NM_001143820) with custom flanking restriction enzyme sites (GENEWIZ, South Plainfield, NJ) was cloned into the pLOC vector (OHS5832; Dharmacon/Horizon Discovery, Cambridge, UK). Lentivirus was produced in HEK-293T cells using either the Trans-Lentiviral ORF Packaging Kit (Dharmacon/Horizon Discovery) or Lipofectamine 3000 (Thermo Fisher Scientific). Viral supernatants were collected 72 hours (hrs) post-transfection, concentrated (PEG Virus Precipitation Kit, Abcam, Waltham, MA), and added dropwise to cells in the presence of media supplemented with 8 μg/ml polybrene. Transduced cells were selected using puromycin (2 μg/ml, Thermo Fisher Scientific). For the generation of CRISPR lines, cells were transduced with virus expressing dCas9-VP64 (Addgene, Watertown, MA), selected using blasticidin (6 – 10 μg/ml, Thermo Fisher Scientific), and subjected to single-cell sorting. Plasmids expressing ETS1 sgRNAs (see Supplemental Table S1) were co-electroporated into the dCas9-VP64-expressing cells using the Amaxa^™ Cell Line Nucleofector^™ Kit R (Lonza, VCA-1001) and further subjected to puromycin selection and single-cell cloning. CRISPR-activation efficiency was assessed by qPCR and/or immunoblotting.

iSLK and iSLK-219 cells (kindly provided by JinJong Myoung) were maintained in DMEM supplemented with 10% fetal bovine serum, L-glutamine (2 mM, Invitrogen), penicillin (100 IU/ml, Gibco) and streptomycin (100 ug/ml, Gibco) at 37°C under a 5% CO₂ atmosphere. iSLK-219 cells were grown in the presence of puromycin (10 mg/ml, Invivogen) to maintain selection for the viral episome. LEC-219 cells were maintained in EBM-2 (Lonza cc3156) media supplemented with the EGM2-MV kit (Lonza cc3203) in presence of 0.25 ug/ml puromycin to maintain selection for the viral episome. BCBL-1 cells were maintained in RPMI supplemented with 10% fetal bovine serum, L-glutamine (2 mM, Invitrogen), penicillin (100 IU/ml, Gibco) and streptomycin (100 ug/ml, Gibco) at 37°C under a 5% CO₂ atmosphere.