Puromicin

HCT 116^p53−/− cells and rpL3ΔHCT 116^p53−/− cells, derived from HCT 116^p53−/− cell line and stably silenced for rpL3, were cultured in Dulbecco's Modified Eagle's Medium (DMEM) with glutamax (Invitrogen, Carlsbad, California) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, penicillin-streptomycin 50 U/ml and 0.5 μg/ml puromycin (Sigma-Aldrich).
CBSΔHCT 116^p53−/− cell line was obtained from HCT 116^p53−/− cells as previously reported [34 (link)]. Cells were transfected with 2 μg of different shRNA CBS plasmids (Sigma-Aldrich) by using Lipofectamin 2000 (Life Technologies) according to the manufacturer's instructions. Stable clones were selected in medium containing 1 mg/ml of Puromicin (Sigma-Aldrich) and assayed for the detection of CBS expression level by western blotting.
shRNA transfections were performed in cells as previously described [34 (link)].
Drug treatments were performed by adding to cells 100 μM 5-FU (Sigma-Aldrich, St. Louis, MO, USA), CHX

To mimic the BRCA1 mutation status HB-2 cells were stably transfected with four different sh-RNAs against BRCA1 containing constructs and a scrambled sh-RNA containing plasmid as a negative control (Qiagen, Hamburg, Germany) by using Lipofectamine 2000^C (Life Technologies Inc., Grand Island, NY, USA). Transfection conditions were calibrated with GFP construct containing plasmid. Selection of transfected cells was done in the presence of 50 ng/ml puromicin (Sigma, Rehovot, Israel) in which 90% of the non-transfected cells are killed. BRCA1 protein level was measured by Western blot in order to choose the best BRCA1 silencing construct. Clone #4 exhibited the highest silencing efficiency (~30%) and was therefore chosen for our experimental system (Supplementary Figure S1). Cells were grown in the presence of 50 ng/ml puromicin. The silencing of BRCA1 was monitored periodically throughout the experiment and found to be stable (not shown). In addition, the proliferation rate, viability and the morphology of the transfected cells revealed no change throughout the whole experimental period compared with the parent clone (not shown).

Human breast adenocarcinoma MCF7 cell line (ATCC^® HTB22™) and human immortalized keratinocytes HaCaT (CLS # 300493) were used. pLKO.1 lentiviral DNA constructs together with the pΔR8.2 (#12263, Addgene) and pVSV-G (#8454, Addgene) packaging plasmids were transfected into 293FT packaging cells (R70007, ThermoFisher) using TurboFect Transfection Reagent (R0531, Thermo Scientific). Virus-containing supernatants were collected 24 to 48 h after transfection and used to infect the recipient cells in the presence of 8 μg/mL polybrene (SIGMA). Infected cell cultures were selected for 4-5 days in medium containing 1 μg/mL puromicin (SIGMA) for pLKO.1-puro constructs. All experiments were performed 5-8 days after vector-mediated gene transfer.