Resistant subclone evasion of CYT387 in vitro. (A) Baf3-EpoR cells expressing JAK2^V617F were subjected to ENU mutagenesis in the presence of CYT387. Cells were plated at 2 × 10⁵ cells per well of a 96 well plate in media with 4μM CYT387. Wells were examined every 5 days for colony outgrowth over a 38-day period. Colonies that grew out were expanded and subsequently serum-starved overnight in the presence of 4μM CYT387 and subjected to immunoblot analysis using antibodies specific for total or phospho-JAK2 or β-actin. (B) Densitometric analysis of immunoblots in panel A. Expression levels for total JAK2 () and phospho JAK2 (■) were normalized to the levels observed in JAK2^WT cells that are sensitive to CYT387. (C) Genomic DNA was isolated from CYT387-resistant Ba/F3 cells and levels of genomic GFP or GAPDH were assessed by quantitative PCR. A standard curve of varying ratios of GFP-positive/GFP-negative Ba/F3 cell mixtures was included as in Figure 5A. Levels of genomic GFP were normalized as in Figure 5 (normalized first to GAPDH, then to the highest value well on the standard curve) and are presented as percent GFP-positive. Values represent mean ± SEM (n = 3).