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C. Fugazza et al.
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Figure 3. Gain/loss of Coup-TFII expression alters the β to γ ratio in opposite directions. Coup-TFII overexpression (OE) was obtained as described in Figure 2; Coup- TFII knockout (KO) was achieved using a CRISPR/Cas9 lentiviral vector and the corresponding empty vectors (EV) were used as a control. (A) Western blot confirming the Coup-TFII KO and OE. U2AF: protein loading control. The Coup-TFII KO was also confirmed by quantitative real-time polymerase reaction RTqPCR (not shown). (B) RTqPCR analysis of individual globin genes in a pool of six single clones at day 60 (KO) and on a pooled population of Coup-TFII OE cells. As a control, a pool of two clones infected with the EV were analyzed. Globin expression levels relative to GAPDH (n≥3, error bars: standard error of mean; *P<0.05) are shown. Right panel: γ- to β-globin ratio (γ+β=1).
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Figure 4. Chromatin immunoprecipitation experiment. (A) Venn diagram illustrating the selection criteria used to identify bona fide Coup-TFII-bound regions. (B) Gene Ontology mouse phenotype association. Erythroid-related phenotypes are shaded in pink. (C) Results of the MEME motif discovery analysis. (D) Pie chart depicting the relative percentages of motifs found by MEME.
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haematologica | 2021; 106(2)