Level of ROS. (A) For evaluation of intracellular ROS level, K562 cells (5 × 105) were washed and incubated for 30 minutes with 50M 2′,7′-dichlorofluorescein-diacetate (DCFH-DA). Cells and analyzed by flow cytometry. Duplicate cells were analyzed before inducing to undergo erythroid differentiation (a representative graph of 2 independent experiments). (B) Cells were analyzed 3 days after induction of erythroid differentiation (a representative graph of 2 independent experiments). (C) Calculation of the fold increase in the dichlorofluorescein fluorescence relative to the control wild-type cells. (D-E) Purified CD34+ from 2 donors were transduced with lentivectors containing either shSBDS-3 or shSCR control, sorted for expressing the YFP reporter gene, and induced erythroid differentiation. The cells were analyzed on day 5 for ROS levels by DCFH-DA. (F) Calculation of the fold increase in the dichlorofluorescein fluorescence relative to the scrambled RNA expressing cells.
