Transfection of human primary adipocytes in suspension with siRNA is associated with decreased expression of targeted proteins.

<p>The human adipocytes were transfected on day 9 post-induction (1.165×10⁵ cells/100 nM siRNA ) and plated at 4.1×10⁴ cells/cm². Forty-eight hours post-transfection, transfection efficiency was assayed as uptake of fluorescent-labeled siRNA and the siRNA-mediated effect on targeted protein expression. (A) Brightfield and fluorescent (rhodamine filter) images of the transfected cells were taken with a 40X objective. (B) Knockdown of lamin A/C and PPARγ was assayed via western blot analysis post-transfection with either the RISC-free control siRNA (control), nontargeting siRNA (NT), or siRNA targeted to the indicated gene (target). Thirty-five µg of protein was loaded in each lane and separated by SDS-PAGE. Equal loading of each lane was determined using β-actin expression. The mean and standard deviation of the ratio lamin A/C and PPARγ compared to β-actin was determined after the expression levels of each protein were quantified using Un-Scan-It software (version 6.1) from samples run in triplicate. The experiment was carried out twice independently.