Retroviral suppression of the let-7 family of miRNAs regulates BCL11A and HbF. (A) Relative expression levels of the let-7 family of miRNAs (open bars represent control, black bars represent LIN28B-OE, and hatch-marked bars represent let-7 sponge) after transduction of the let-7 sponge or LIN28B-OE encoding retrovirus. The qRT-PCR analyses were performed at culture day 14, and compared with control transductions. The miRNAs relative expression levels (y-axis) in the control cells were defined as a level of one for comparison. Error bars denote ± standard deviation of 3 independent donors for each condition. (B) Western blot analyses with protein extracts from the empty vector control (C) versus LIN28B-OE (OE) and let-7 sponge (SP) cells. The membranes were probed with anti-BCL11A. Anti-β-actin probe was used as a loading control. HPLC analyses of hemoglobin from (C) control, (D) LIN28B-OE, and (E) let-7 sponge samples were performed at culture day 21. HbF and HbA peaks are labeled on each graph (y-axis: mVolts; x-axis: elution time in minutes). Data are representative of 3 independent experiments. P values were calculated using two-tailed Student t test. C, empty vector control; OE, LIN28B overexpression; SP, let-7 sponge. *P < .05.