HMGB2 expression increases and the knockdown of HMGB2 impedes Gfi-1B expression during erythroid differentiation. (A) HMGB2 protein expression during erythroid differentiation. CD34⁺ cells were purified from cord blood samples and cultured in a 2-phase system. During the first phase of 5 days (D1 to D5), CD34⁺ cells were cultured in the presence of IL-3, SCF, EPO, and dexamethasone. Then, the cells were induced to differentiate in the presence of EPO and SCF (E0 to E5). Cell lysates were prepared from CD34⁺ cells before (D3) and every day after induction of erythroid differentiation (E0 to E5). Total cell extracts were separated by SDS-PAGE and analyzed by Western blotting using an HMGB2-specific antibody. Actin expression was evaluated to confirm equal protein loading. Data are also expressed (bottom part of the figure) as the ratio between HMGB2 and actin proteins. (B) Experimental protocol for cell transduction and analysis of the effects of HMGB2 depletion. CD34⁺ cells were purified from cord blood and amplified in the presence of IL-3, SCF, EPO, and dexamethasone and then infected with lentiviruses carrying shControl or shHMGB2 and the puromycin-resistance gene (D1 and D2). Cells were then grown for an additional 48 hours in liquid culture in the culture medium but with addition of 1 μg/mL puromycin. After puromycin selection, cells were induced to differentiate in the presence of EPO and SCF (E0 to E5). (C) Gfi-1B expression during erythroid differentiation in the presence or absence of HMGB2. Cell lysates were prepared from shControl- or HMGB2-transduced cells the day of the induction of erythroid differentiation (E0) and 3 (E3) or 5 (E5) days after. Proteins were analyzed by Western blot using specific antibodies as indicated on the left of the figure; 3 independent experiments were performed.