Figure 2.
Figure 2. STAT5 and Bcl-xL are implicated in EEC formation. (A) Inhibition of Bcl-xL and STAT5 in primary cells using an siRNA strategy decreased CFU-E formation. Day-5 CD36+ cells cultured in the presence of Epo, DXM, IL-3, and SCF were electroporated with siRNA targeted on either STAT5 or Bcl-xL, or with a nonspecific control sequence. GpA- cells were plated at day 6 in methylcellulose in the presence of Epo and SCF. CFU-Es were counted 7 days later. Number of CFU-Es was significantly reduced after knock-down of either STAT5 or Bcl-XL compared with the control (n = 3; Student t test: P < .05). (B-C) A constitutively active form of STAT5 as well as Bcl-xL overexpression induced EEC formation in methylcellulose assays. PB CD34+ cells were cultured in the presence of Epo, SCF, IL-3, and DXM, transduced at days 4 and 5 either with the MIGR, STAT5CA, or Bcl-xL vectors. At day 7, CD36+/GpA-/GFP+ cells were sorted, and 5000 cells were plated in methylcellulose in the presence of SCF alone. As a positive control, 1000 cells were plated in parallel in the presence of SCF and Epo. Histograms represent the total number of CFU-Es at day 7. In the presence of Epo, the CFU-E number was higher with the STAT5CA and the Bcl-xL vectors than with the control (B). In the absence of Epo (C), whereas MIGR-transduced cells did not give rise to a significant number of CFU-Es, either STAT5CA or Bcl-xL vectors could induce EEC formation (n = 3, each in triplicate; Student t test: STAT5CA vs MIGR, P < .05; Bcl-xL vs MIGR, P < .05). Data are represented as mean ± SEM. (D) Qualitative differences between STAT5CA-induced (left) and Bcl-xL-induced (right) EECs. Bcl-xL-induced EECs were not only less numerous (Student t test: P = .001), but also contained a lower number of cells than the STAT5CA-induced CFU-Es. These Bcl-xL-induced EECs were very similar to those routinely observed in PV patients. CFUs were counted using a Zeiss Telaval 31 microscope (Zeiss, Oberkochen, Germany) and a 20×/0.35 numeric aperture objective (Micromecanique, Evry, France). Images were captured using a Nikon Eclipse TE300 microscope (Nikon, Tokyo, Japan) connected to a Zeiss Axiocam digital camera. Images were acquired using Zeiss Axiovision 4 software.

STAT5 and Bcl-xL are implicated in EEC formation. (A) Inhibition of Bcl-xL and STAT5 in primary cells using an siRNA strategy decreased CFU-E formation. Day-5 CD36+ cells cultured in the presence of Epo, DXM, IL-3, and SCF were electroporated with siRNA targeted on either STAT5 or Bcl-xL, or with a nonspecific control sequence. GpA- cells were plated at day 6 in methylcellulose in the presence of Epo and SCF. CFU-Es were counted 7 days later. Number of CFU-Es was significantly reduced after knock-down of either STAT5 or Bcl-XL compared with the control (n = 3; Student t test: P < .05). (B-C) A constitutively active form of STAT5 as well as Bcl-xL overexpression induced EEC formation in methylcellulose assays. PB CD34+ cells were cultured in the presence of Epo, SCF, IL-3, and DXM, transduced at days 4 and 5 either with the MIGR, STAT5CA, or Bcl-xL vectors. At day 7, CD36+/GpA-/GFP+ cells were sorted, and 5000 cells were plated in methylcellulose in the presence of SCF alone. As a positive control, 1000 cells were plated in parallel in the presence of SCF and Epo. Histograms represent the total number of CFU-Es at day 7. In the presence of Epo, the CFU-E number was higher with the STAT5CA and the Bcl-xL vectors than with the control (B). In the absence of Epo (C), whereas MIGR-transduced cells did not give rise to a significant number of CFU-Es, either STAT5CA or Bcl-xL vectors could induce EEC formation (n = 3, each in triplicate; Student t test: STAT5CA vs MIGR, P < .05; Bcl-xL vs MIGR, P < .05). Data are represented as mean ± SEM. (D) Qualitative differences between STAT5CA-induced (left) and Bcl-xL-induced (right) EECs. Bcl-xL-induced EECs were not only less numerous (Student t test: P = .001), but also contained a lower number of cells than the STAT5CA-induced CFU-Es. These Bcl-xL-induced EECs were very similar to those routinely observed in PV patients. CFUs were counted using a Zeiss Telaval 31 microscope (Zeiss, Oberkochen, Germany) and a 20×/0.35 numeric aperture objective (Micromecanique, Evry, France). Images were captured using a Nikon Eclipse TE300 microscope (Nikon, Tokyo, Japan) connected to a Zeiss Axiocam digital camera. Images were acquired using Zeiss Axiovision 4 software.

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