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. Author manuscript; available in PMC: 2018 Jan 23.
Published in final edited form as: Exp Hematol. 2016 Aug 31;44(12):1138–1155.e4. doi: 10.1016/j.exphem.2016.08.007

Figure 3. Effect of increasing concentrations of SB431542 on the expansion of progenitor (A) and precursor (B-D) cells in HEMA cultures of MNC from Adult Blood with (bottom panel) or without (top panel) Dex.

Figure 3

Figure 3

A) Cultured cells were collected at 0, 3, 6 and 10 Days of HEMA cultures of AB containing increasing concentrations of SB431542 (0, 1, 3, 10 and 26 μM) and their progenitor cell content evaluated as colony forming cells (CFC) in semisolid cultures. The columns present the total CFC numbers and the grey area within the columns the numbers of CFU-E-, BFU-E- and CFU-GM-derived colonies. Results are presented as Mean of those obtained in three separate experiments, each one with a different donor, performed in duplicate. SEMs are not indicated for clarity. Statistical analysis was performed by Student’s t-test and the results are summarized in Fig. S1.

B) Cells generated by MNC from AB cultured in HEMA in the presence of increasing concentrations of SB431542 (0, 1, 3, 10 and 26 μM) over time (0, 3, 6, 8, 10, 12, 14 and 17 Days). Results are presented as fold increase with respect to Day 0 and are presented as Mean (± SEM) of three experiments each one with a different donor. Statistical analysis was performed by mixed models and Student’s t-tests and the results are summarized in Fig. S1.

C) Representative flow cytometric analyses for CD36 and CD235a expression of cells from AB cultured for 17 Days in the absence or presence of Dex and treated with or without SB431542 (26 µM), as indicated. Erys were recognized and divided into distinctive maturation stages on the basis of standard criteria based on the levels of CD36pos/CD235aneg expression indicated by rectangles [32, 33]. Events indicated with light blue, blue, red and grey dots represent non-erythroid cells, proErys, basophilic/polychromatic Erys and orthochromatic Erys, respectively. The numbers near each rectangle indicate the frequency of the different populations within the analyzed population.

D) May-Grunwald staining of cytospin preparations of Erys obtained at Day 14 and 17 of HEMA from AB with and without Dex in the presence of SB431542 (0, 10 or 26 μM), as indicated. The arrows in D indicate cells in metaphase. Results are representative of those observed in four experiments, one of which seeded with purified CD34+ cells. Original magnification 200x.