Figure 1
Figure 1. Bcl-xL is up-regulated in association with AE expression and is critical for the survival of AE cells. (A) CD34+ UCB cells were transduced with vector- or AE-expressing retroviruses, followed by lysis for RNA extraction and quantitative PCR for Bcl-xL and Bcl-2. c-abl gene expression was used for normalization. (B) CD34+ UCB cells were transduced with vector (C) or AE-expressing retrovirus, followed by lysis for protein extraction and immunoblotting at indicated time points. (C) CD34+ and CD34− AE cells were separated using magnetic beads, followed by lysis for protein extraction and Western blotting. (D) CD34+ UCB or AE cells were transduced with NT shRNA, Bcl-xL shRNA2, or Bcl-xL shRNA3. The percentage of transduced AE cells was determined by flow cytometry at days 3, 5, and 7. The percentages at days 5 and 7 were normalized to day 3 within each group, followed by normalizing each time point in the Bcl-xL shRNA2 and Bcl-xL shRNA3 groups to the NT shRNA group. Data represents mean ± standard deviation (SD; n = 4;*P < .05). (E) Scatter plots showing percent change in Venus(+) cells at week 6 compared with initial transduction efficiency at day 3. Total BM cells were obtained by flushing the injected bone with PBS using a 28-G needle, followed by red blood cell lysis and labeling of the remaining cells with antibody against human CD45 surface antigen for flow cytometry analysis. Each dot represents 1 mouse. Bars indicate mean. (F) CD34+ AE/pBabe or AE/Bcl-2 cells were transduced with NT shRNA, Bcl-xL shRNA2, or Bcl-xL shRNA3. The percentage of transduced AE cells was determined by flow cytometry at indicated time points, followed by normalization as described in panel D. Data represents mean ± SD (n = 5; *P < .05). (G) Left: shRNA-transduced AE cells were fixed and permeabilized at day 3, followed by intracellular labeling with antibody against active caspase 3 for flow cytometry analysis. Right: shRNA-transduced AE cells were labeled with annexin-V and 7-AAD at day 3 for flow cytometry analysis. Data represents Mean ± SD (n = 3; *P < .05). (H) CD34+ AE/pBabe or AE/Bcl-2 cells were transduced with NT shRNA, Bcl-xL shRNA2, or Bcl-xL shRNA3. Transduced cells were sorted, followed by labeling with annexin V and 7-AAD for flow cytometry analysis. Data represents Mean ± SD (n = 3; *P < .05; NS indicates nonsignificant).

Bcl-xL is up-regulated in association with AE expression and is critical for the survival of AE cells. (A) CD34+ UCB cells were transduced with vector- or AE-expressing retroviruses, followed by lysis for RNA extraction and quantitative PCR for Bcl-xL and Bcl-2. c-abl gene expression was used for normalization. (B) CD34+ UCB cells were transduced with vector (C) or AE-expressing retrovirus, followed by lysis for protein extraction and immunoblotting at indicated time points. (C) CD34+ and CD34− AE cells were separated using magnetic beads, followed by lysis for protein extraction and Western blotting. (D) CD34+ UCB or AE cells were transduced with NT shRNA, Bcl-xL shRNA2, or Bcl-xL shRNA3. The percentage of transduced AE cells was determined by flow cytometry at days 3, 5, and 7. The percentages at days 5 and 7 were normalized to day 3 within each group, followed by normalizing each time point in the Bcl-xL shRNA2 and Bcl-xL shRNA3 groups to the NT shRNA group. Data represents mean ± standard deviation (SD; n = 4;*P < .05). (E) Scatter plots showing percent change in Venus(+) cells at week 6 compared with initial transduction efficiency at day 3. Total BM cells were obtained by flushing the injected bone with PBS using a 28-G needle, followed by red blood cell lysis and labeling of the remaining cells with antibody against human CD45 surface antigen for flow cytometry analysis. Each dot represents 1 mouse. Bars indicate mean. (F) CD34+ AE/pBabe or AE/Bcl-2 cells were transduced with NT shRNA, Bcl-xL shRNA2, or Bcl-xL shRNA3. The percentage of transduced AE cells was determined by flow cytometry at indicated time points, followed by normalization as described in panel D. Data represents mean ± SD (n = 5; *P < .05). (G) Left: shRNA-transduced AE cells were fixed and permeabilized at day 3, followed by intracellular labeling with antibody against active caspase 3 for flow cytometry analysis. Right: shRNA-transduced AE cells were labeled with annexin-V and 7-AAD at day 3 for flow cytometry analysis. Data represents Mean ± SD (n = 3; *P < .05). (H) CD34+ AE/pBabe or AE/Bcl-2 cells were transduced with NT shRNA, Bcl-xL shRNA2, or Bcl-xL shRNA3. Transduced cells were sorted, followed by labeling with annexin V and 7-AAD for flow cytometry analysis. Data represents Mean ± SD (n = 3; *P < .05; NS indicates nonsignificant).

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