Fig. 3.
Fig. 3. Increased survival in FKBP51 overexpressing cells. / (A) UT-7/Mpl cell survival in cytokine-free medium. UT-7/Mpl cells were infected with a retrovirus encoding both FKBP51 and EGFP or a control vector containing EGFP alone. EGFP+ cells were sorted and permanent cell lines were obtained in the presence of GM-CSF. Cells were deprived in GM-CSF and living cells were counted at day 2 by trypan blue exclusion test or MTT test. Results from the trypan blue exclusion test are presented. EGFP+ cells and the wild-type UT-7/Mpl cells have similar survival, whereas FKBP51 overexpressing UT-7 stay alive for up to 5 days after cytokine deprivation (P = .0035). (B) Increased survival of UT-7/Mpl overexpressing FKBP51 is related to a resistance to apoptosis. UT-7/Mpl expressing EGFP and UT-7/Mpl overexpressing FKBP51 cells (UT-7/Mpl-M51) were grown in the presence of GM-CSF. Cytokine deprivation was performed for 36 hours, and binding of annexin V was assessed by flow cytometry. Cells dead by apoptosis (positive for both 7-AAD and annexin V) were markedly reduced in UT-7/Mpl-M51 cells (4%) in comparison to UT-7/Mpl-EGFP cells (16.5%). (C) Inhibition of procaspase-3 cleavage by overexpression of FKBP51. Western blots were performed in UT-7/Mpl expressing EGFP and UT-7/Mpl cells overexpressing FKBP51 cultured with GM-CSF or without growth factor for 24 hours. Cleavage of procaspase-3 was noticed in normal cells after cytokine withdrawal (lane 2) but not in UT-7/Mpl cells overexpressing FKBP51 (lane 4). (D) UT-7/Mpl cell survival in GM-CSF after daunorubicin treatment. UT-7/Mpl expressing EGFP and UT-7/Mpl overexpressing FKBP51 cells (UT-7/Mpl-M51) were grown in the presence of GM-CSF and treated by 5 μM daunorubicin. Cell survival was evaluated 24 hours later (P = .023). (E) CD34+ cell survival in serum-free cytokine-free medium. Normal CD34+ cells were transduced by retroviral vector encoding both FKBP51 and EGFP or EGFP alone after 48 hours of stimulation by cytokines. Cells were then cultured in serum-free medium at 50 000 cells/mL. Cell survival was evaluated by counting cells each day using the trypan blue exclusion test. Results are the average of 3 experiments (day 3,P = .083; day 4, P = .027; day 7,P = .023).

Increased survival in FKBP51 overexpressing cells.

(A) UT-7/Mpl cell survival in cytokine-free medium. UT-7/Mpl cells were infected with a retrovirus encoding both FKBP51 and EGFP or a control vector containing EGFP alone. EGFP+ cells were sorted and permanent cell lines were obtained in the presence of GM-CSF. Cells were deprived in GM-CSF and living cells were counted at day 2 by trypan blue exclusion test or MTT test. Results from the trypan blue exclusion test are presented. EGFP+ cells and the wild-type UT-7/Mpl cells have similar survival, whereas FKBP51 overexpressing UT-7 stay alive for up to 5 days after cytokine deprivation (P = .0035). (B) Increased survival of UT-7/Mpl overexpressing FKBP51 is related to a resistance to apoptosis. UT-7/Mpl expressing EGFP and UT-7/Mpl overexpressing FKBP51 cells (UT-7/Mpl-M51) were grown in the presence of GM-CSF. Cytokine deprivation was performed for 36 hours, and binding of annexin V was assessed by flow cytometry. Cells dead by apoptosis (positive for both 7-AAD and annexin V) were markedly reduced in UT-7/Mpl-M51 cells (4%) in comparison to UT-7/Mpl-EGFP cells (16.5%). (C) Inhibition of procaspase-3 cleavage by overexpression of FKBP51. Western blots were performed in UT-7/Mpl expressing EGFP and UT-7/Mpl cells overexpressing FKBP51 cultured with GM-CSF or without growth factor for 24 hours. Cleavage of procaspase-3 was noticed in normal cells after cytokine withdrawal (lane 2) but not in UT-7/Mpl cells overexpressing FKBP51 (lane 4). (D) UT-7/Mpl cell survival in GM-CSF after daunorubicin treatment. UT-7/Mpl expressing EGFP and UT-7/Mpl overexpressing FKBP51 cells (UT-7/Mpl-M51) were grown in the presence of GM-CSF and treated by 5 μM daunorubicin. Cell survival was evaluated 24 hours later (P = .023). (E) CD34+ cell survival in serum-free cytokine-free medium. Normal CD34+ cells were transduced by retroviral vector encoding both FKBP51 and EGFP or EGFP alone after 48 hours of stimulation by cytokines. Cells were then cultured in serum-free medium at 50 000 cells/mL. Cell survival was evaluated by counting cells each day using the trypan blue exclusion test. Results are the average of 3 experiments (day 3,P = .083; day 4, P = .027; day 7,P = .023).

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