Page 170 - 2021_06-Haematologica-web

Z. Shah et al.
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Figure 5. MYB inactivation leads to a severe deficiency of primitive clonogenic progenitors. (A) Morphology of individual CFU-C colonies derived from day 6 and day 10 hematopoietic progenitors (upper panels; scale bar, 100 mm), and May-Grünwald staining of cells from corresponding colonies (lower panels; scale bar, 20 mm). MΦ: macrophages; MK: megakaryocytes; E: erythroblasts; Gr: granulocytes. (B) Real-time polymerase chain reaction (RT-PCR) analysis of hemoglobin gene expres- sion. Differentiated H1 embryonic stem cells (ESC) (1x104 cells) were grown in methylcellulose assay medium for 18 days, and all resulting CFU-C colonies (~100- 150) were pooled for mRNA isolation. The data are mean ± standard deviation (SD). (C) Primitive human clonogenic progenitors express MYB. The frequency of the hematopoietic progenitors was measured in SKO1 CD34+Venus+ versus CD34+Venus– cells. Data are mean ± SD, n=3. (D) The frequency of clonogenic hematopoietic progenitors in total or non-adherent cells of the three H1-isogenic MYB genotypes. Mean values ± SD are shown, n=3. *P<0.05; NS: non-significant; two-tailed Student’s t-test. (E) Morphology of typical DKO erythroid colonies and cells. Upper panels, scale bar – 100 mm; lower panels, scale bar – 20 mm. (F) Morphology of typical DKO myeloid colonies and cells. Scale bar – 20 mm. (G) Activin stimulates MYB-Venus expression in the MYB knockout cell lines on day 6 of differentiation. Data are mean ± SD, n=4. *P<0.05; two-tailed Student’s t-test. (H) Activin stimulation of day 6 primitive clonogenic progenitors of the three H1-isogenic MYB geno- types. Data are mean ± SD, n=3. *P<0.05; NS: non-significant; two-tailed Student’s t-test.
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haematologica | 2021; 106(8)