1. NF-κB superrepressor was overexpressed by means of the bicistronic retrovirus.
NF-κB superrepressor was overexpressed by means of the bicistronic retrovirus. Infection of UT7 by the NF-κBsr retroviral vector. A, UT7 eGFP and UT7 FKBP51 cells were infected with the retrovirus containing the entire coding region of NF-κBsr. This retrovirus encodes the same reporter gene (eGFP) as the previous retroviral vectors, which had been used to obtain these cell lines. Therefore, we selected <5% of cells with the highest intensity of eGFP. Permanent cell lines were obtained. UT7 Mpl cells were also infected as a control for infection. B, nuclear and cytoplasmic extracts of proteins from UT7 eGFP and UT7 FKBP51 cells with or without NF-κBsr were submitted to Western blot analysis for NF-κB subunits and IκBα expression, respectively. Arrows, positions of endogenous and exogenous IκBα. C, transcriptional activity of NF-κB was analyzed in UT7 eGFP and UT7 FKBP51 cells with or without NF-κBsr. Cells were transiently transfected with NF-κB luciferase reporter gene plasmids. After being cultured with GM-CSF during 20 hours, cells were lysed and assayed for reporter gene activity. Luciferase activities were normalized to relative luciferase unit (RLU) per μg of protein. *., P < D, cells were washed with PBS thrice and cultured with or without GM-CSF. After 24 hours, the ratio of apoptotic cells was analyzed after phycoerythrin/Annexin V and 7-amino-actinomycin D (7-AAD) staining by flow cytometry. E, percentages of apoptotic cells. Cell survival/proliferation rate was also analyzed by MTT assay after 48 hours. *, P <
Emiko Komura et al. Cancer Res 2005;65:
©2005 by American Association for Cancer Research