Blasticidin

HEK293T cells were cotransfected with pCMV R8.2 (addgene, #12263), pMD2.G (addgene, #12259) and pLenti6V5A-AltB2RFlag (or the empty vector for Mock cell line) using Lipofectamine 2000 as per the manufacturer’s instructions. After 48 h at 37 °C, the cell media was filtered through 0.45 μM pores and the viral supernatant collected. Polybrene (1 μg/ml) and the viral supernatant (2 ml) were added to HeLa cells grown in 6-well plates, and the cells were incubated at 37 °C for 48 h. The media was then replaced with media containing 2 μg/ml of blasticidin (Wisent, #450-190-XL), and the cells were incubated at 37 °C for another 24 h. Cells were then passaged while gradually increasing the blasticidin concentration at every passage (2, 4, 8, 15 μg/ml). Stable cell lines were maintained in DMEM containing 10% FBS, 1% antimicrobial/antimycoplasma and 15 μg/ml of blasticidin. Stable cells from a 6-well plate were washed with PBS, scraped with RIPA (50 mM Tris-HCl, pH 7.5, 0.1% SDS, 1% Sodium Deoxycholate, 1% Triton X-100, complete protease inhibitors (Roche, #11873580001)), and sonicated. Samples were quantified using BCA protein assay reagent and 100 μg of protein was diluted in Laemmli (βME). Samples were boiled for 5 min at 95 °C and processed for western blot analysis as described above with anti-AltB2R (1/1000; 1:1 stock solution).

The stable U2OS cell lines expressing myc-BirA∗-DCAF, myc-BirA∗-DDB1, and GFP-DDB1 were generated using the Flp-In T-Rex system (Thermo Fisher Scientific) using respectively pGLAP1-myc-BirA∗-DCAF, pGLAP1-myc-BirA∗-DDB1, and pGLAP1-GFP-DDB1 constructions. U2OS Flp-In-Rex (U2OS-FT) cells were maintained at 37 °C, 5% CO₂ in Dulbecco’s Modified Eagle Medium (Thermo Fisher Scientific), supplemented with 10% FB essence (Wisent, St John’s), 50 U/ml penicillin/streptomycin, and 10 mM Hepes. U2OS-FT cells were transfected using Lipofectamine LTX (Thermo Fisher Scientific) during 48 h in 6 cm Petri dishes with 4.5 μg of Flp-Recombinase expression vector pOG44 (Thermo Fisher Scientific) and 500 ng of plasmid DNA. Transfected cells were selected for 2 weeks with hygromycin (50 μg/ml, Thermo Fisher Scientific) and blasticidin (10 μg/ml, Wisent). The expression of the cDNA was achieved by adding 10 μg/ml doxycycline (Clontech Laboratories, Mountain View) in the medium for 24 h or 48 h (or not as control). Cells were lysed directly in Laemmli sample buffer, and the extracted proteins were resolved by SDS-PAGE prior to being transferred to a nitrocellulose membrane. Immunoblotting was performed with a BirA∗ antibody (Novus Biologicals #6C4c7, 1:1000 dilution) or GFP (Santa Cruz Sc-9996, 1:1000 dilution). To inhibit cullins, we treated cells 24 h with 10 μM MLN4924 (Cell signaling 85923S).

Pairs of gRNAs flanking the CREs of interest, FOXA1 promoter and control regions were designed using CRISPOR (http://crispor.tefor.net/) and Zhang lab CRISPR Design tool (http://crispr.mit.edu/) (Supplementary Data). Each pair of gRNAs were cloned into the lentiCRISPRv2 (Addgene; a gift from Feng Zhang #52961) and the lentiCRISPRv2-Blast (Addgene; a gift from Feng Zhang #83480) plasmid as previously described^{69 (link)}. Lentiviral particles were generated in 293FT cells (ThermoFisher) using the pMDG.2 and psPAX2 packaging plasmids (Addgene; #12259 and #12260, a gift from Didier Trono), and collected 72 hrs post transfection. LNCaP cells were transduced for 24–48 hrs with equal amounts of virus, followed by selection with media containing puromycin (3.5 µg/mL, ThermoFisher) and blasticidin (7 µg/mL, Wisent). Cells were harvested upon selection for RNA and DNA for RT-PCR and confirmation of DNA cleavage, respectively. For cell proliferation, cells were seeded at equal density per well (on a 96-well plate; Day 1) upon puromycin and blasticidin selection. Growth of the cells were monitored by cell counting using Countess automated cell counter (Invitrogen). Cell numbers were calculated as a percentage compared to negative control. Statistical significance was calculated using Student’s t test.