Figure 4
Figure 4. KLF4 infected cells arrest in G1 and die by apoptosis. (A) v-Abl cells were infected with MIC or MIC-KLF4 and then pulsed with BrdU for 6 hours before harvesting at 48 hours. Cells were stained with anti-BrdU antibody and 7-AAD to assess cell-cycle status. Top panel includes the percentages of each phase of the cell cycle, with BrdU-positive cells boxed. Bottom circular gates represent, from left to right, subdiploid, G1, combined S/G2 that have not divided during the BrdU pulse. The bottom panel shows the forward–side scatter dot plot of the G1 cells that have not entered S phase during the BrdU pulse. Many cells within this gate were no longer live cells based on laser scatter parameters and percent death is displayed above the gate. (B) BCR-ABL cells, before or after infection with MIG-Bcl-XL, were superinfected with MIC-KLF4 or empty MIC virus. Some KLF4-transduced samples were treated with zVAD-fmk (100 μM), which was not toxic under these conditions (data not shown). Cells were stained for hCD4 at 24 hours or 48 hours and then analyzed by FACS. The percentage of hCD4+ cells remaining from 24 hours to 48 hours (maintenance) was calculated as in Figure 2A. Error bars represent standard deviations of 3 independent experiments. (C) BCR-ABL cells were treated with etoposide (20 μM) or infected with MIC-KLF4, in the absence or presence of Bcl-XL overexpression, as indicated. Staining with 7-AAD, annexin V, and DioC6 was used to assess plasma-membrane integrity, phosphatidylserine exposure, and mitochondrial-membrane potential. (D) BCR-ABL cells infected with empty MIC virus (CD4) or MIC-KLF4 were treated as in panel C then fixed, permeabilized, and stained for cleaved caspase-3. Data are representative of at least 3 experiments. (E) Cells were infected with MIC-KLF4 and/or MIG-Bcl-XL, then cultured in the absence or presence of etoposide (concentrations in μM) for 24 hours before sorting for hCD4 marker gene expression. Equivalent numbers of sorted cells were then immunoblotted with indicated antibodies, and β-actin was used as a loading control.

KLF4 infected cells arrest in G1 and die by apoptosis. (A) v-Abl cells were infected with MIC or MIC-KLF4 and then pulsed with BrdU for 6 hours before harvesting at 48 hours. Cells were stained with anti-BrdU antibody and 7-AAD to assess cell-cycle status. Top panel includes the percentages of each phase of the cell cycle, with BrdU-positive cells boxed. Bottom circular gates represent, from left to right, subdiploid, G1, combined S/G2 that have not divided during the BrdU pulse. The bottom panel shows the forward–side scatter dot plot of the G1 cells that have not entered S phase during the BrdU pulse. Many cells within this gate were no longer live cells based on laser scatter parameters and percent death is displayed above the gate. (B) BCR-ABL cells, before or after infection with MIG-Bcl-XL, were superinfected with MIC-KLF4 or empty MIC virus. Some KLF4-transduced samples were treated with zVAD-fmk (100 μM), which was not toxic under these conditions (data not shown). Cells were stained for hCD4 at 24 hours or 48 hours and then analyzed by FACS. The percentage of hCD4+ cells remaining from 24 hours to 48 hours (maintenance) was calculated as in Figure 2A. Error bars represent standard deviations of 3 independent experiments. (C) BCR-ABL cells were treated with etoposide (20 μM) or infected with MIC-KLF4, in the absence or presence of Bcl-XL overexpression, as indicated. Staining with 7-AAD, annexin V, and DioC6 was used to assess plasma-membrane integrity, phosphatidylserine exposure, and mitochondrial-membrane potential. (D) BCR-ABL cells infected with empty MIC virus (CD4) or MIC-KLF4 were treated as in panel C then fixed, permeabilized, and stained for cleaved caspase-3. Data are representative of at least 3 experiments. (E) Cells were infected with MIC-KLF4 and/or MIG-Bcl-XL, then cultured in the absence or presence of etoposide (concentrations in μM) for 24 hours before sorting for hCD4 marker gene expression. Equivalent numbers of sorted cells were then immunoblotted with indicated antibodies, and β-actin was used as a loading control.

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