JAK2 stimulates HR in Ba/F3 cell lines. (A) HR substrate (HR-EGFP/3′EGFP) contains a tandem repeat of 2 inactive EGFP genes, HR-EGFP and 3′EGFP. When the I-SceI endonuclease is expressed, a DSB is introduced at the I-SceI site in the HR-EGFP gene. Recombination restores a functional EGFP gene, and the recombinant cells can be monitored by fluorescence detection methods. Importantly, because the 3′EGFP cassette is only deleted at the 5′ end, EGFP fluorescence not only monitors gene conversion events associated with or without crossing over, but also single strand annealing. (B) Ba/F3-EPOR cells were electroporated with HR substrate, and the puromycin-resistant colonies were isolated on methylcellulose. Genomic DNA was prepared and intrachromosomal integration was verified by PCR as detailed in “Cell lines.” (C,D) Ba/F3-HR2 were infected with the empty retroviral vector or viruses encoding JAK2wt, JAK2^V617F, or TEL-JAK2. (C) JAK2 protein levels were analyzed by Western blotting using an anti-JAK2 antibody or (D) STAT5 phosphorylation was detected after EPO stimulation (10 U/mL) at various times by Western blotting analysis using an anti-[pY⁷⁰⁵]STAT5 antibody. (E) GFP⁺ cells were measured by flow cytometry analysis to determine HR. Percentages of GFP⁺ cells are given as the means plus or minus SD of at least 6 independent experiments. (F) Cells were infected or not with a lentivirus encoding I-SceI, and induced HR was calculated as the difference between the percentage of GFP⁺ cells infected with I-SceI and the percentage of GFP⁺ cells noninfected for each condition. Results are means plus or minus SD of 4 independent experiments. *Statistical significance (P < .05) using Student t test.