Figure 1
Overexpression of Bcl11b inhibits proliferation of the hematopoietic progenitor cell line FDC-P1 but has no effect on apoptosis. (A) Expression of Bcl11b in FDC-P1 and cells transfected with the mutated versions K828T, S778N, Y844C and FS823 was detected by Western blot and RT-PCR. In the Western blot analysis, the human cell line Jurkat was included as a positive control. The two bands correspond to different isoforms of Bcl11b. (B) Cell viability was determined by staining with propidium iodide followed by flow cytometric analysis. Data shown are the mean ± SD (n = 6). (C) Proliferation of non-transfected FDC-P1 cells and FDC-P1 cells transfected with wild-type Bcl11b or the mutated versions (K828T, S778N or Y844C) was measured by [3H]-TdR incorporation (cpm × 10-3) after 72 hours of SCF-stimulation. Results are presented as mean ± SD (n = 3). FDCP1: 11.8 ± 1.7; wild-type Bcl11b: 4.4 ± 1.1; K828T: 37.5 ± 3.3; S778N: 19.3 ± 1.9; Y844C: 32.4 ± 0.9. (D) Cells were stained with CFSE and cultured for 72 hours in SCF, after which CFSE fluorescence was determined by flow cytometry. Histograms for the four different mutants K828T, S778N, Y844C and FS823 (K, S, Y and FS, respectively) are shown. CFSE fluorescence declines (shift to the left on the X-axis) as cell numbers increase. In each histogram, data from non-transfected FDC-P1 cells (F) and cells transfected with wild-type Bcl11b (wt) are overlaid for a comparison. Results are from one representative experiment of three performed.