Page 66 - Haematologica April 2020

P. Balogh et al.
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Figure 2. RUNX3 participates in erythroid but not granulocytic differentiation of human progenitors. (A) Flow cytometry plots for erythroid differentiation of CD34+ cells transduced with empty vector (EV) or RUNX3-targeting (RUNX3 sh4) lentiviral shRNA constructs and subjected to unilineage erythroid culture for three days. Graph summarizes the quantitation of erythroid differentiation from three independent experiments. (B) Summary of colony formation assays on CD34+ cells trans- duced as in (A). (C) Flow cytometry histogram overlays for granulocyte differentiation of CD34+ cells transduced as in (A) and subjected to unilineage granulocytic cul- ture for eight days. Representative results from three independent experiments. (D) Flow cytometry histogram overlays for cell proliferation by dye dilution. Transduced CD34+ cells were stained with PKH26 followed by three days of culture in expansion medium (CD34) or erythroid medium. Graph summarizes mean flu- orescence intensity due to dye retention from three independent experiments. (E) Summary of viability as assessed by flow cytometry on CD34+ cells transduced as in (A) and cultured in expansion or erythroid medium for indicated days. (F) Flow cytometry plots for erythroid differentiation of CD36+ CD235a– early committed ery- throid progenitors transduced as in (A) and cultured in erythroid medium for three days. Graph summarizes quantitation of erythroid differentiation from three inde- pendent experiments. (G and H) Summary of hemoglobinization, i.e. percent cells positive for benzidine staining, in HUDEP-2 cells transduced with control vectors (EV), RUNX3-knockdown lentivirus (sh4), or RUNX3-overexpression retrovirus (OE). Cells were cultured 48 hours in HUDEP-2 expansion (Uninduced) or differentiation (Induced) medium. N=3. (A and C) Two-tailed Student t-test. (B, D, and F-H) Two-way ANOVA with Bonferroni’s test. (E) One-way ANOVA with Tukey’s test. *P<0.05; **P<0.01; ***P<0.005. Error bars+standard error of mean.
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haematologica | 2020; 105(4)