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. 2013 May 6;210(5):1003–1019. doi: 10.1084/jem.20120521

Figure 4.

Figure 4.

NF-E2 mutations confer a proliferative advantage on the MPN clone by enhancing cell proliferation. (A–D) Mononuclear cells of the MPN patients indicated were seeded in methylcellulose and individual erythroid and myeloid colonies harvested after 14 d. Colonies were assayed for presence of the JAK2V617F and the NF-E2 mutations by BsaXI restriction of PCR-amplified DNA (Baxter et al., 2005) and by GeneScan analysis (patients 202, 209, and 532) or direct sequencing (patients 209 and 409), respectively. Genotypes of the individual colonies, each represented by a single dot, are depicted. (E) FDCP-1-EpoR cells (left) and BaF3 cells (right) carrying the JAK2V617F mutation were transduced either with a vector expressing the 262aa NF-E2 truncation mutant or with an empty control vector. Total cell extracts were interrogated with an antibody that recognizes both human and murine NF-E2. Equal loading was assured by reprobing with an antibody against GAPDH. (F and G) FDCP-1-EpoR cells (F) and BaF3 cells (G) carrying the JAK2V617F mutation and expressing either the 262aa NF-E2 truncation mutant or carrying an empty control vector were cultured in the absence of growth factors (left) or in the presence of 1 IU/ml Epo or 5 ng/ml IL-3, as indicated. Mean and SEM of four independent experiments are shown. **, P < 0.01; ***, P < 0.001. (H) FDCP-1-EpoR cells carrying an empty control vector (left) or expressing the 262aa NF-E2 truncation mutant (right) were stained with Hoechst 33342 and analyzed for DNA content by FACS. Representative blots of n = 6 independent experiments are shown. (I) Statistical analysis of n = 6 independent experiments. *, P < 0.05; **, P < 0.01 by Student’s t test. (J) Cell extracts of FDCP-1-EpoR cells carrying an empty control vector (left lanes) or expressing the 262aa NF-E2 truncation mutant (right lanes) were interrogated with antibodies against CDK4, CDK6, and cyclin D3, as indicated. Equal loading was assured by reprobing with an antibody against β-actin. Representative blots of n = 6 independent experiments are shown. (K) Statistical analysis of n = 6 independent Western blot experiments. Mean and SEM are shown. *, P < 0.05 by Wilcox matched pairs test.