Nuclear Transcription Factor Detection.

Cellular fate is determined by the activity of nuclear transcription factors. Here, we describe a series of protocols for detecting transcription factors at both the transcript and protein levels in human adipose cells. Methods for analysis of transcript include RNA extraction, reverse transcription polymerase chain reaction (RT-PCR), endpoint PCR, and RT-qPCR. Evaluation of protein expression includes protocols for protein extraction, quantification by Bradford assay, SDS-PAGE, western blotting, and quantification with ImageJ. Each of these steps is critical for a reliable and reproducible assessment of transcription expression and characterization of human adult-derived adipose stromal/stem cells.

MED31 involved in regulating self-renewal and adipogenesis of human mesenchymal stem cells.

Regulation of gene expression is critical for the maintenance of cell state and homeostasis. Aberrant regulation of genes can lead to unwanted cell proliferation or misdirected differentiation. Here we investigate the role of MED31, a highly conserved subunit of the Mediator complex, to determine the role this subunit plays in the maintenance of human mesenchymal stem cell (hMSC) state. Using siRNA-mediated knockdown of MED31 we demonstrate a decrease in self-renewal based on cell assays and monitoring of gene expression. In addition, in the absence of MED31, hMSCs also displayed a reduction in adipogenesis as evidenced by diminished lipid vesicle formation and expression of specific adipogenic markers. These data present evidence for a significant role for MED31 in maintaining adult stem cell homeostasis, thereby introducing potential novel targets for future investigation and use in better understanding stem cell behavior and adipogenesis.