Figure 3.
Analysis of β0-HUDEP-2 differentiation following transduction with the LVβ-shα2 lentiviral vector
(A) Morphological changes of HUDEP-2 and β0-HUDEP-2 cells following differentiation. Day 0, day 4, and day 8 samples were stained with May-Grünwald Giemsa. Arrows indicate advanced stages of erythroid differentiation (Ortho-E, Poly-E, and Baso-E are indicated on day 4 and day 8). Magnification: 1,000×; scale bars, 25 μm. (B) Erythroid progenitor populations on day 0, day 4, and day 8 of differentiation. Cells at different stages of differentiation were counted from images taken at 400× magnification from cytospin slides. Percentages of different erythroid populations (Ortho-E, Poly-E, and Baso-E) for day 4 and day 8 of differentiation are shown (n = 2, >200 cells counted per cytospin). (C) Hemoglobin variant analysis by HPLC of the soluble cellular fraction obtained from HUDEP-2 and β0-HUDEP-2 cells transduced with BB305, LVβ-shSCR, and LVβ-shα2 on day 10 of erythroid differentiation. (D) Cumulative cell index of HUDEP-2, β0-HUDEP-2, and β0-HUDEP-2 cells transduced with the BB305, LVβ-shSCR, and LVβ-shα2 vectors over a 10 day time-course of erythroid differentiation. Data are presented as mean ± SD of two independent experiments. (E) Relative α2/α1-globin mRNA expression levels in HUDEP-2, β0-HUDEP-2, and β0-HUDEP-2 cells transduced with the BB305, LVβ-shSCR, and LVβ-shα2 vectors. (F) Western blot analysis of soluble and membrane fractions of HUDEP-2, β0-HUDEP-2, and transduced β0-HUDEP-2 cells after 10 days of erythroid differentiation. The antibody used for β-globin detection cross-reacts with δ-globin and therefore contributes to the faint band observed with β0-HUDEP-2 cells.