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. Author manuscript; available in PMC: 2015 Jul 14.
Published in final edited form as: Sci Transl Med. 2014 May 28;6(238):238ra71. doi: 10.1126/scitranslmed.3008004

Fig. 4. Effect of BRafV600E mutation on HSPC differentiation, self-renewal, and GC response to alloantigen.

Fig. 4

(A and B) Plating of whole BM (A) and sorted LMPP cells (B) in methylcellulose medium containing myeloid and erythroid cytokines (EPO, IL-3, IL-6, and SCF) or IL-7. BRafV600E cells could be replated for >10 platings in the presence of IL-7. Photograph of initial plating shown on left. (C and D) GC response in Cd19-cre BRafV600E (n = 5) and control mice (n = 5) 10 days after SRBC injection by gross photographs of mouse spleens (top), flow cytometric assessment (bottom and bar graph on right) (C), and immunohistochemistry for peanut agglutinin (PNA) (D). Scale bars, 100 μm. C, Cre-negative BRafV600E control; KI, Cd19-cre BRafV600E. (E and F) GC response in Cd19-cre BRafV600E and control mice alongside age-matched mice with GC-restricted BRafV600E expression (Cγ1-cre BRafV600E) by flow cytometry (E) and by PNA stain (F). Scale bars, 500 μm (top) and 100 μm (bottom). (G and H) Competitive transplantation of Mx1-cre BRafV600E (n = 10 recipient mice) compared with Cre-negative BRafV600E whole BM cells (n = 10 recipient mice) 4 weeks (G) and up to 16 weeks (H) after transplantation. (I) Mice transplanted with BRafV600E hematopoietic cells in a competitive manner (n = 10 mice in control and n = 10 mice in knock-in group) developed anemia and thrombocytopenia concomitant with expansion of engrafted BRafV600E HSPCs as shown in (H). Error bars represent means ± SD for (A) to (C), (E), and (H). Bar represents mean value in (I). *P < 0.05 (Mann-Whitney U test).