Puromycin

After the 72 h transfection period in 2250 μl in six-well plates, MCF10A cells were treated with an additional 750 μl of medium containing 3 μM puromycin (Mirus Bio 5940), achieving a final concentration of 1 μM (0.5 μg/ml) puromycin and final volume of 3 ml (54–55 (link),71 (link),73 (link)). Cells were incubated 1 h at 37°C, then washed with cold 1× PBS. Protein was isolated and analyzed as above.

Fresh healthy bone marrow samples were purchased from Lonza (1M-105, Lonza). Whole bone marrow was incubated with ammonium chloride (07800, StemCell Technologies) for 10 min on ice for lysis of red blood cells followed by a wash with PBS until obtaining a pellet of white cells. CD34⁺ cells were enriched using the Miltenyi kit (19056, Miltenyi Biotech) and following the manufacturer’s instructions.
Isolated human CD34⁺ cells were cultured in non-tissue culture treated plates for 36 hours in StemSpan SFEMII medium (09655, Stem Cell Technologies) with 100ng/ml of the following recombinant human cytokines: SCF (300–07, Peprotech), TPO (300–18, Peprotech), Flt3 (300–19, Peprotech) and IL-6 (200–06, Peprotech). Cells were then transferred to non-TC 96-well plates in a viral prep consisting of fresh media supplemented with polybrene (TR-1003-G, Sigma) and a lentivirus producing a shRNA against human FANCD2; a MOI of 50 was used for the scrambled shRNA and a MOI of 100 was used for the FANCD2 shRNAs. Plates were spun down at 2300 rpm for 30 minutes at RT and incubated for 12–16 hours. Selection media with 1 μg/ml puromycin (MIR 5940, MirusBio) was added to cultures 12–24 hours after viral infection. FA-like cells were selected in this puromycin-containing media for 72 hours.