Off-target effects dominate a large-scale RNAi screen for modulators of the TGF-β pathway and reveal microRNA regulation of TGFBR2.

Background: RNA interference (RNAi) screens have been used to identify novel components of signal-transduction pathways in a variety of organisms. We performed a small interfering (si)RNA screen for novel members of the transforming growth factor (TGF)-β pathway in a human keratinocyte cell line. The TGF-β pathway is integral to mammalian cell proliferation and survival, and aberrant TGF-β responses have been strongly implicated in cancer.

Probing lipid rafts with proximity imaging: actions of proatherogenic stimuli.

Glycosylphosphatidylinositol (GPI)-anchored proteins have been shown to cluster in microdomains enriched in glycosphingolipids and cholesterol and represent a relatively selective marker of lipid rafts. In recent years, several attempts have been made to use fluorescent probes to nondisruptively label these domains in living cells. Here, we have transfected endothelial cells with a GPI-anchored thermotolerant green fluorescent protein (ttGFP) to show colocalization of this fluoroprobe with another marker of lipid rafts, urokinase-type plasminogen activator receptor-1.

Sequence characteristics of functional siRNAs.

RNA interference in mammalian cells is actively used to conduct genetic screens, to identify and to validate targets, and to elucidate regulators and modifiers of cellular pathways. To ensure the specificity and efficacy of the active 21mer siRNA molecules, it is pertinent to develop a strategy for their rational design. Here we show that most functional siRNAs have characteristic sequence features.