Hygromycin

rKSHV.219 was multiplied by the induction of iSLK.219 cells with doxycycline as previously described (Myoung and Ganem, 2011 (link)). Briefly, iSLK.219 cells were induced in both the presence of doxycycline (Sigma, United States) and the absence of hygromycin, puromycin, and G418 (Sigma, United States) at approximately 90% confluency. Four days post-induction, cell supernatants were collected, cleared twice by centrifugation (2,500 × g, 30 min at 4°C), virus was passed through 0.45 μm pore size membrane filters, and then pelleted by ultracentrifugation (25,000 × g, 3 h at 4°C) using a CP-80WX (Hitachi, Japan). Virus pellets were then resuspended overnight in serum-free DMEM collection media and stored at −80°C. KSHV titer (GFU/mL) was determined in HEK293T cells by counting GFP-positive cells as described previously (Zhao et al., 2015 (link)).
Titer (GFU/mL) = [(green fluorescent cells/field)] × [(fields/well)/volume virus (mL) × (dilution factor)].

Packaging of the pQCXIH and pQCXIH-ISceI retroviral vectors and infection of cell cultures was performed as previously described (40 (link)). The selection for cells infected with pQCXIH-ISceI was achieved by growth in medium containing 50 μg/ml hygromycin (Sigma) for 14 or 15 days, with medium changes every 2 days to allow for expression of I-SceI endonuclease and the generation of DSBs. After 13 days, the cells were trypsinized and replated. After an additional 1 or 2 days, the cells were trypsinized again, pooled, and either analyzed for the frequency of GFP-positive and DsRed-positive cells, or used for isolation of genomic DNA for analysis of small deletions.

Yeast pre-cultures were done by selecting a colony from cultures on fresh yeast extract-peptone-dextrose (YEPD) agar plates (10 g/L Bacto-yeast extract, 20 g/L Bacto-peptone and 20 g/L dextrose). Sporulation was induced on spoMA medium (1 g/L Bacto-yeast extract, 0.5 g/L glucose, 10 g/L potassium acetate and 20 mg/L adenine) at 28 °C. Transformed strains were selected and grown on YEPD medium supplemented with 200 mg/L geneticin (Sigma-Aldrich, Lezennes, France), hygromycin (Sigma-Aldrich) or nourseothricin (Sigma-Aldrich)^{63 (link)}. Flocculation degree was quantified in YNB+glucose (YNB 0.67% w/v, glucose 2% w/v), YEPD and synthetic must MS425^{64 (link)}. Cells were stored in YEPD broth supplemented with glycerol (Sigma-Aldrich, 20% v/v final concentration) at −80 °C.

The retroviral vector pBABEpuro containing either EGFR-WT, EGFR-G719S (gifts from H. Greulich), or EGFR-E746-A750del or no insert (empty vector) (all confirmed by sequencing) were transfected into the amphotropic packaging cell line, Phoenix (ATCC), using Lipofectamine 2000 (Invitrogen). After 48 h viral supernatant was harvested, filtered (45 μm) and used to infect MCF10A cells in the presence of 8 μg/mL polybrene (Sigma-Aldrich). Cell lines were selected with 1 μg/mL puromycin for one week (Sigma-Aldrich) and designated MCF10A EGFR-WT, MCF10A EGFR-GS and MCF10A EGFR-DEL, respectively. To aid in assessing tumour formation assays, MCF10A and MCF10CA1a cells were tagged with luciferase using the MSCV Luciferase PGK-hygromycin (Addgene) retroviral vector. Vector DNA was transfected into Phoenix cells as described above. Virus was harvested 48, 54, 72 and 78 post-transfection and used in sequential infection of the MCF10CA1a cells (total of 4 infections) with 8 μg/ml polybrene. Stable transfectants were selected with the addition of 200 μg/ml hygromycin (Sigma-Aldrich). The luciferase-tagged cell lines were then infected with the EGFR-WT, EGFR-G719S, EGFR-E746-A750del vectors and designated MCF10CA1a- EGFR-WT, EGFR-GS or EGFR-DEL, accordingly. MCF10CA1a cells infected with empty pBABEpuro retroviral vector (EV) were used as controls (MCF10A and MCF10CA1a-EV).