Figure 2.

Efficient knockdown of BCL11A and ZNF410 by double shmiR vector in erythrocytes differentiated in vitro from transduced gene-marked SCD patient CD34⁺ HSPCs

(A) BCL11A and ZNF410 mRNA expression was measured by qRT-PCR with GAPDH as control on day 11 of differentiation. Data represent mean ± SD, n = 3. (B) Induction of γ-globin mRNA was determined on day 18 of differentiation by qRT-PCR. Data represent mean ± SD, n = 3, ∗p < 0.05. (C) HbF of cell lysates was measured by HPLC on day 18 of differentiation. Data represent mean ± SD, n = 3. ∗p < 0.05. (D) Cell proliferation of shmiR transduced cells during erythroid differentiation. Data represent mean ± SD, n = 3. (E) Phase-contrast microscope image of representative sample of Hoechst 33342⁻ sorted enucleated erythroid progeny 30 min after sodium MBS treatment from erythrocytes differentiated from nontransduced or transduced SCD CD34⁺ HSPCs; white arrows indicate sickle forms; scale bar, 50 μm. (F) Quantification of sickled cells from nontransduced and transced enucleated erythroid cells at 30 min after MBS treatment. Data are plotted as means, symbols indicate different SCD patients, and each data point represents an independent replicate. ∗∗p < 0.01. (G) Correlation of HbF expression assessed by HPLC versus numbers of sickled cells. Black dots represent samples transduced with different shmiR vectors or nontransduced. Correlation coefficient (r²) is shown for all data.