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Fig. 5. Ectopic expression of GFP-survivin in HEK293T cells reversed the reduction in cell proliferation and changes in the cell cycle caused by caveolin-1. (A) HEK293T cells were transfected with pLacIOP alone (3 µg) or co-transfected with the vectors pLacIOP-caveolin-1 (3 µg) and increasing amounts of the vector pEGFP-survivin (up to 2 µg). Controls included transfections with pEGFP-C1 (2 µg) or pEGFP-survivin (2 µg). Cell proliferation was measured by the MTS® assay. Values detected following transfection are shown as a percentage of the MTS activity measured in non-transfected controls (100%). (B) HEK293T cells were either transfected with pEGFP-caveolin-1 alone (5 µg) or co-transfected with the vectors pEGFP-caveolin-1 (5 µg) and pEGFP-survivin (5 µg) and cell cycle was analyzed by flow cytometry 48 hours post-transfection by gating on GFP-negative (lane 1) and GFP-positive (lanes 2 and 3) subpopulations. GFP-caveolin-1 and GFP-survivin expression are shown in a representative western blot. A result representative of two independent experiments is shown. (C) HEK293T cells were transfected with the vector pLacIOP (3 µg) alone or co-transfected with the vectors pLacIOP-caveolin-1 (3 µg) and increasing amounts of pEGFP-survivin (up to 2 µg) and cultured post-transfection in the presence of either 250 nM doxorrubicin, 25 µM etoposide or 250 nM taxol. Cell proliferation was evaluated by the MTS® assay. Values shown are equivalent to the percentage of residual activity as compared with non-transfected controls (100%). In A and C the data shown are mean ± s.e.m. from three independent experiments; *comparison with pLacIOP control, P<0.01; #comparison with pLacIOP-caveolin-1, P<0.01.
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