Figure 1.

Efficient knockdown of BCL11A and ZNF410 by double shmiR vector leads to high γ-globin and HbF induction in erythroid cells differentiated in vitro from transduced gene-marked human CD34⁺ HSPCs

(A) Schematic of virus transduction and erythroid differentiation of human CD34⁺ HSPCs. (B) BCL11A and ZNF410 mRNA expression as measured by qRT-PCR with GAPDH as control on day 11 of differentiation. (C) BCL11A and ZNF410 protein expression as measured by western blot with GAPDH as a loading control on day 11 of differentiation. Densitometric quantitation is shown below each lane. (D) Induction of γ-globin mRNA as determined by qRT-PCR on day 18 of differentiation. (E) HbF of cell lysates as measured by HPLC on day 18 of differentiation. (F) Correlation of γ-globin determined by qRT-PCR versus HbF by HPLC. Black dots represent samples transduced with different shmiR vectors. The Pearson correlation coefficient (r²) is shown. (G) Differentiation status of erythroid cells after 18 days in culture using CD71 and CD235a. (H) Summary of the data shown in (G) normalized to the percentage of the CD71-CD235a⁺ population. (I) Enucleation of in vitro differentiated erythroid cells. (J) Summary of the data shown in (H) normalized to the percentage of the Hoechst⁻ population. Data represent means ± SD, n = 3. ns, not significant; ∗p < 0.05.