Altered expression of globin subunits in KLF1^+/E325K cells. (A) Expression of (i) HBG, (ii) HBB, (iii) HBA, (iv) HBE, (v) HBZ, and (vi) HBD via qPCR. (B) Analysis of β-globin (β), δ-globin (δ), ^Gγ-globin (^Gγ), α-globin (α), and ^Aγ-globin (^Aγ) abundance in WT KLF1^+/E325K, and KLF1^+/− cells on day 6 of differentiation via reverse-phase high-performance liquid chromatography (RP-HPLC); (i) Representative chromatograms and (ii) quantification of chromatogram peak areas (mAU×min; mean ± SD, n = 3; ∗P < .05; ∗∗∗P < .001). (C) Western blots of WT, KLF1^+/E325K, and KLF1^+/− cells probed with antibodies of β-, γ-, and α-globin, with β-actin as a loading control (representative of 3 biological repeats). (D) Total globin abundance in WT, KLF1^+/E325K, and KLF1^+/− cells on day 6 of differentiation determined via RP-HPLC as in panel Bii (mean ± SD; n = 3). (E) Expression of BCL11A in WT, KLF1^+/E325K, and KLF1^+/− cells during expansion (day 0) and on days 2, 4, and 6 of differentiation, determined via qPCR (mean ± SEM; n = 3; ∗P < .05, ∗∗P < .01, and ∗∗∗P < .001 for WT vs KLF1^+/E325K; ⁺P < .05, ⁺⁺P < .01, and ⁺⁺⁺P < .001 for WT vs KLF1^+/−).