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Fig. 3. Caveolin-1 expression in HEK293T cells reduced survivin mRNA and protein content. HEK293T cells were transfected with the plasmids pEGFP-C1, pEGFP-caveolin-1, pLacIOP or pLacIOP-caveolin-1 24 hours prior to measurements. (A) Cell extracts were analyzed by western blotting with anti-survivin, anti-caveolin-1 and anti-actin antibodies. From left to right: non-transfected cells (lane 1) and cells transfected with pEGFP-C1 (lane 2), pEGFP-caveolin-1 (lane 3), pLacIOP (lane 4) or pLacIOP-caveolin-1 (lane 5). Survivin levels were quantified by scanning densitometric analysis of western blots and normalized to actin. Residual survivin protein levels for pEGFP-caveolin-1 (0.55±0.06) and pLacIOP-caveolin-1 (0.49±0.05) transfected cells were compared to their respective mock controls. Numerical data represent the means ± s.e.m. of results obtained in three independent experiments; *comparison with their respective mock controls, P<0.001. (B) Alternatively, survivin was detected by immunofluorescence followed by FACS analysis in both green (GFP-positive) and non-green (GFP-negative) HEK293T cells, after transfection with the pEGFP-caveolin-1 vector. Data are representative of two experiments. (C) RT-PCR analysis of survivin. Actin was used as internal control. From left to right, samples from cells transfected with pEGFP-C1 (lane 1), pEGFP-caveolin-1 (lane 2), pLacIOP (lane 3) or pLacIOP-caveolin-1 (lane 4). Survivin mRNA levels for pEGFP-caveolin-1 (0.59±0.07)- and pLacIOP-caveolin-1 (0.50±0.07)-transfected cells were compared to their respective mock controls. Numerical data represent the means ± s.e.m. of results obtained in three independent experiments; *comparison with their respective mock controls, P<0.05.
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