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. 2013 Jan 24;8(1):e54723. doi: 10.1371/journal.pone.0054723

Figure 2. Effect of AMG531 on MK development.

Figure 2

MKs were differentiated from human umbilical cord blood progenitors and cultured for 13 days in presence of AMG531 at different concentrations. 10 ng/mL of rHuTPO was used as control. (A) Immunofluorescence images of MKs cultured with rHuTPO 10 ng/mL (i) or AMG531 100 ng/mL (ii), 1000 ng/mL (iii) and 2000 ng/mL (iv) at day 13 of culture. Cell samples were citospun on 12 mm cover slips and stained with anti-CD61 antibody (green). Nuclei were counterstained with Hoechst 33288 (blue). Images were acquired through an Olympus BX51, magnification 63×. Scale bar = 25 µm. (B) Western blot analysis of MK differentiation markers CD61, RUNX-1, NFE2. Samples were probed with anti β-actin antibody to demonstrate equal loading. (C) Ploidy levels of MKs was analyzed by flow cytometry as described in Design an Methods. Data are expressed as mean ± SD of 3 different experiments. *p<0.05, ** p<0.01. (D) MK output was calculated as the percentage of CD61+ cells at day 13 of culture and normalized to the total number of CD34+ cells at the beginning of the cell culture (values in parenthesis represent rHuTPO or AMG531 concentrations, expressed as ng/mL). The error bars represent the mean ± SD of 6 independent experiments. *p<0.05, ** p<0.01, ***p<0.001. (E) Immunofluorescence images of proplatelet formig-MKs derived from cultures treated with rHuTPO 10 ng/mL (i) or AMG531 100 ng/mL (ii), 1000 ng/mL (iii) and 2000 ng/mL (iv) stained with anti α-tubulin antibody (green). Nuclei were stained with Hoechst 33288 (blue). Images were acquired through an Olympus BX51, magnification 20×. Scale bar = 50 µm. (F) Histogram shows proplatelet formation (PPF) analyzed after 16 hour adhesion on fibrinogen (values in parenthesis represent rHuTPO or AMG531 concentrations, expressed as ng/mL). The error bars represent the mean ± SD of 5 independent experiments. ***p<0.001. (G) Representative images of proplatelet revealed with anti α-tubulin staining (green). Nuclei were counterstained with Hoechst 33288 (blue). Images were acquired through an Olympus BX51, magnification 63×. Scale bar = 20 µm. (H) Western blot analysis of pAKT, pERK1/2 and pp38 in MKs at day 13 of culture. AKT, ERK1/2, p38 and β-actin were revealed to show equal loading.