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. 2014 Nov 17;211(12):2439–2454. doi: 10.1084/jem.20141123

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© 2014 Ye et al.

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Figure 2. — Deficiency in CCP6 and not in CCP1 causes abnormal megakaryopoiesis. (A and B) Paraffin sections from spleen and femurs of 6-wk-old mice were stained with H&E or anti-VWF antibody (brown)/hematoxylin (blue) by IHC. Bar, 100 µm. Green arrows in left panels and red arrows in right panels denote MKs. MKs in different fields (n = 20 fields) were enumerated (right graphs). Results are shown as means ± SD. ***, P < 0.001. (C) Flow cytometry analysis of HSCs (Lin⁻Sca-1⁺c-Kit⁺/LSK) and myeloid progenitors (Lin⁻c-Kit⁺Sca-1⁻) from BM. (D) Flow cytometry analysis of CLPs (Lin⁻CD127⁺Sca-1^lowc-Kit^low) and CMPs (Lin⁻c-Kit⁺Sca-1⁻CD34⁺CD16/32⁻) from BM. (E) Flow cytometry analysis of MEPs (Lin⁻c-Kit⁺Sca-1⁻CD34⁻CD16/32⁻) and GMPs (Lin⁻c-Kit⁺Sca-1⁻CD34⁺CD16/32⁺) from BM. (F) Flow cytometry analysis of MKPs (Lin⁻c-Kit⁺CD41⁺) and MKs (Lin⁻c-Kit⁻CD41⁺) from BM. n = 10 for each group. Results are shown as means ± SD. ***, P < 0.001. (G) Paraffin sections of BM-containing MKs were stained for AchE by IHC (left). AchE⁺ cells represent all MKs. Red arrows denote MKs. Data are representative of at least three separate experiments. Bar, 20 µm. Total numbers of AchE⁺ cells were enumerated in the right graph (n = 20 fields). Results are shown as means ± SD. ***, P < 0.001. (H) In vitro MK development assays from sorted BM MKPs using MethoCult semisolid media for CFU-MK formation (top left) and liquid suspension media for large size MK maturation (bottom left). Representative microscopy images are shown (left). Bar, 100 µm. Red arrows in top left denote CFU-MK colony and black arrows in bottom left denote large size MKs. 1 × 10² sorted MKPs from BM of the indicated mice were mixed with 1 ml MethoCult media and plated on a 35-mm dish for incubation. MK colonies were enumerated and the colony types of MKs were confirmed by AchE staining. CFU-MK assays from total BM cells (5 × 10⁴) were also performed and displayed similar results (not depicted). Data are representative of at least three separate experiments. n = 5 for each group. Cell numbers were enumerated as specified (right graphs). Results are shown as means ± SD. **, P < 0.01.