1. Differential roles of cAMP and cGMP in megakaryocyte maturation and platelet biogenesis 
Antonija Jurak Begonja, Stepan Gambaryan, Harald Schulze, Sunita Patel-Hett, Joseph E. Italiano, John H. Hartwig, Ulrich Walter 
Experimental Hematology 
Volume 41, Issue 1, Pages e4 (January 2013)
DOI: /j.exphem
Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

2. Figure 1
Increased expression of β-sGC, PKG-I, PKA-C, VASP, and MENA during differentiation of mouse FLC into MKs. (A) FLC and MKs separated from non-MK cells after 2 and 4 days in culture and mouse platelets (Plt) were analyzed for expression levels of endothelial NOS (eNOS), β-sGC, PKG-I, PKA-C (catalytic subunit), MENA, VASP, and phospho-VASP (Ser157, Ser239) by Western blotting. Human umbilical vein endothelial cells were used as a control for eNOS and mouse brain lysate as a control for MENA expression. GAPDH was used as a loading control. Blots representative of four independent experiments are shown. (B) Proplatelets contain PKG-I, VASP, and MENA. Immunofluorescence images of MKs with proplatelets stained for PKG-I, VASP, MENA, or β-tubulin. The images are representative of four independent experiments. (C) Colocalization of VASP phosphoforms in proplatelets. Immunofluorescence images of proplatelets stained with mouse anti–phospho-VASP Ser157 and rabbit anti–phospho-VASP Ser239 representative of three independent experiments are shown. Scale bar: 5 μm.
Experimental Hematology  , e4DOI: ( /j.exphem )
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3. Figure 2
cAMP and cGMP levels and PDEs expression changes during MK development. (A) cAMP and (B) cGMP levels were measured in FLC, day 2 and 4 MK, non-MK cells, and mouse platelets (Plt) by electroimmunoassay. The results, normalized to cell proteins, are representative of three independent experiments and are means ± standard deviation. ∗A t test significance of p < 0.05 compared to FLC at day 0, n = 3. (C) Expression levels of PDE2A, PDE3A, PDE4A1, and PDE5 were analyzed by Western blotting in FLC, MK, non-MK cells after 2 and 4 days in culture, and mouse platelets. Representative blots from three independent experiments are shown. (D) PKG and PKA stimulation in MK cultures. Gradient purified MKs were preincubated for 1 hour in serum-free Dulbecco's modified Eagle medium without TPO and then treated for 5 min with sodium nitroprusid (SNP; 10 μM), 8-pCPT-cGMP (25 μM), forskolin (5 μM), or PGE1 (1 μM). Anti–phospho VASP Ser239 antibody was used to analyze PKG and PKA activation. Ser157 is a PKA preferential phosphorylation site that shifts VASP mobility from 46 to 50 kDa. Therefore two bands, or a shifted band, indicate phosphorylation of both sites. PKG-I was used as a loading control. Blots representative of three independent experiments are shown.
Experimental Hematology  , e4DOI: ( /j.exphem )
Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions

4. Figure 3
The cAMP, but not cGMP, enhances MK development. (A) To modulate the cAMP pathway, FLC were cultured with TPO and treated daily with forskolin (1 and 5 μM) and PGE1 (1 μM). Cultures were analyzed on day 4 for proliferation (total cell count [i]), percent CD61 expressed (ii), and for counts of CD61+ cells (iii). (B) Expression of PKA-C, VASP, phospho-VASP 157Ser, phospho-VASP 239Ser, and GAPDH (loading control) in MKs. (C) To modulate the cGMP pathway, FLC were cultured with TPO, stimulated daily with ODQ (10 μM), DEA/NO (5 μM), L-NAME (100 μM), and 8-pCPT-cGMP (100 μM), and analyzed on day 4 for total cell counts (i), percent CD61 expressed (ii), and counts of CD61+ cells (iii). (D) Expression of β-sGC, PKG-I, VASP, phospho-VASP 157Ser, phospho-VASP 239Ser, and GAPDH, as loading controls, were evaluated in MKs. ∗Statistically significant differences compared to control; p < 0.05, n = 6.
Experimental Hematology  , e4DOI: ( /j.exphem )
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5. Figure 4
PGE1 and forskolin inhibit MK polyploidization. FLC were cultured with TPO, stimulated daily with forskolin (5 μM), PGE1 (1 μM), or 0.01% ethanol, as a solvent delivery control. The ploidy of MKs on day 4 was assessed by propidium iodide staining. (A) Histograms representative of three experiments are presented. (B) Ploidy is expressed as fold increase relative to control 2N, mean ± standard deviation of three experiments. ∗Significance of p < 0.05 compared to the ploidy of the control, n = 4.
Experimental Hematology  , e4DOI: ( /j.exphem )
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6. Figure 5
Opposite effects of PKG and PKA on platelet formation. (A) Platelets released from the MK cultures (i) were identified by the presence of microtubular coils, the defining characteristic of peripheral blood platelets (ii, β-tubulin staining). (B) Gradient purified day 3 MKs were cultured overnight in the absence or presence of DEA/NO (5 μM), 8-pCPT-cGMP (100 μM), BAY (1 μM), forskolin (5 μM), or PGE1 (1 μM). Platelet production was analyzed by flow cytometry, gating platelet-sized CD61+ particles. Data are presented as the fold increase in platelets released from the untreated MKs (defined as 1). ∗Significance of p < 0.05 as compared to the control, n = 4. (C) VASP phosphorylation at Ser239 after 24 hours incubation with indicated substances.
Experimental Hematology  , e4DOI: ( /j.exphem )
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7. Supplementary Figure E1
(A) Expression of CD41 and CD61 in MK cultures. Fetal livers from 13.5 days postcoital embryos were explanted and cultured in Dulbecco's modified Eagle medium with TPO (10 ng/mL) for 4 days. Cultures were analyzed by FACS for CD41 and CD61 expression (days 0 to 4). FLC are CD41+ on day 0. Expression of CD61 gradually increases as MKs mature in culture. Results are presented as mean ± standard deviation from three independent experiments. (B) Characterization of FLC, MKs, or non-MKs after 2 and 4 days in culture. FLC, MKs (cells collected in pellet after BSA gradient), and non-MKs (nonsedimented cell fraction of gradient) from day 2 and 4 cultures were analyzed by phase-contrast microscopy and FACS (CD61+). The starting FLC and non-MKs consisted of small cells that were negative for CD61 or have 4% to 6% CD61+ cells, respectively. Gradient enriched MKs are large cells, 70% to 80% and 80% to 90% CD61+ after days 2 and 4, respectively. (C) FLC were 2N to 4N. Day 2 MKs were ≤32N. Ploidy on day 4 increased to 64N.
Experimental Hematology  , e4DOI: ( /j.exphem )
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8. Supplementary Figure E2
(A, B) MK cAMP and cGMP levels are not affected by TPO. Day 4 enhanced MKs were treated with forskolin (5 μM, 5 min), sodium nitroprusid (SNP; 10 μM, 5 min), or TPO (10 ng/mL, 15 min) and cAMP or cGMP levels measured by electroimmunoassay. Results of three independent experiments were normalized to the protein concentration and are means ± standard deviation. ∗Significance of p < 0.05, compared to control, n = 3, t test. (C) PKA/PKG activation does not interfere with TPO/c-Mpl signaling. Day 4 enriched MKs were resuspended in serum-free media, starved for 1 hour, and then stimulated for 15 min with TPO (10 ng/mL), 8-pCPT-cGMP (25 μM), or forskolin (5 μM) in the presence or absence of TPO. Phosphorylation of JAK2 (JAK2-P, Tyr 1007/1008) or VASP (VASP-P, Ser 239) was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blotting in MKs. Total JAK2 and VASP were used as loading controls. Representative blots from three independent experiments are shown.
Experimental Hematology  , e4DOI: ( /j.exphem )
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9. Supplementary Figure E3
(A) Signal densities from Figure 3B were quantified and expression levels of PKA-C, VASP, P-VASP Ser157, and P-VASP Ser239 in MKs (day 4) after treatment of cell cultures with PGE1 (1 μM) and forskolin (1 μM, 5 μM) are shown. (B) Signal densities from Figure 3D were quantified and expression levels of β-sGC, PKG-I, VASP, P-VASP Ser157, and P-VASP Ser239 in MKs (day 4) after treatment of cell cultures with ODQ (10 μM), DEA/NO (5 μM), L-NAME (100 μM), and 8-pCPT-cGMP (100 μM) are shown. Each bar represents the relative expression of the proteins indicated after different treatments, and setting the value from control as 1. ∗Statistically significant compared to control; p < 0.05, analysis of variance.
Experimental Hematology  , e4DOI: ( /j.exphem )
Copyright © 2013 ISEH - Society for Hematology and Stem Cells Terms and Conditions