Apoptosis in Bcl11a-deficient B cell precursors could be rescued by exogenous Bcl2. (A) Apoptosis was measured by flow cytometry in Bcl11a-deficient early B cells in the BM. Numbers in histograms are percentages of the indicated gates. (B) Western blot analysis of Bcl11a protein at indicated time points after Tam treatment. (C) Apoptosis in pro–B cells (IMDM supplemented with 50 ng/ml of IL-7, Flt3L, and SCF) after Bcl11a deletion detected by Annexin V and 7-AAD. Numbers in the flow cytometry plots refer to percentages of gated cells. (D) 1 million cultured pro–B cells were treated with Tam for 4 d, and live cells were enumerated at indicated time points. (E) Detection of intracellular Bcl2 in cultured pro–B cells at indicated time points after Tam treatment. (F) Apoptosis in cultured pro–B cells infected with retrovirus-expressing Bcl2. GFP⁺ cells (infected) were gated for analysis. An empty retroviral vector expressing GFP was used as the control. (G) qRT-PCR analysis of key lymphoid genes in cultured Bcl11a-deficient pro–B cells. Data represent mean values of three independent biological replicates and all values are normalized to Gapdh expression. Error bars indicate the SD. (H) Two-way hierarchical clustering of expression of 100 important lymphoid genes (Table S4) in cultured pro–B cells. Expression of these genes in wild-type DN3 thymocytes was used as an expression control. Scale indicates the log2 value of normalized signal level. For A, C, D, E, F, and G, data represent at least three independent experiments.