Efficient and Cost Effective Electroporation Method to Study Primary Cilium-dependent Signaling Pathways in the Granule Cell Precursor.

The primary cilium is a critical signaling organelle found on nearly every cell that transduces Hedgehog (Hh) signaling stimuli from the cell surface. In the granule cell precursor (GCP), the primary cilium acts as a pivotal signaling center that orchestrates precursor cell proliferation by modulating the Hh signaling pathway. The investigation of primary cilium-dependent Hh signaling machinery is facilitated by in vitro genetic manipulation of the pathway components to visualize their dynamic localization to the primary cilium. However, transfection of transgenes in the primary cultures of GCPs using the currently known electroporation methods is generally costly and often results in low cell viability and undesirable transfection efficiency. This paper introduces an efficient, cost-effective, and simple electroporation protocol that demonstrates a high transfection efficiency of ~80-90% and optimal cell viability. This is a simple, reproducible, and efficient genetic modification method that is applicable to the study of the primary cilium-dependent Hedgehog signaling pathway in primary GCP cultures.

The development of a novel transforming growth factor-ß (TGF-ß) inhibitor that disrupts ligand-receptor interactions.

Transforming growth factor-ß (TGF-ß) plays an important role in regulating epithelial to mesenchymal transition (EMT) and the TGF-ß signaling pathway is a potential target for therapeutic intervention in the development of many diseases, such as fibrosis and cancer. Most currently available inhibitors of TGF-ß signaling function as TGF-ß receptor I (TßR-I) kinase inhibitors, however, such kinase inhibitors often lack specificity. In the present study, we targeted the extracellular protein binding domain of the TGF-ß receptor II (TßR-II) to interfere with the protein-protein interactions (PPIs) between TGF-ß and its receptors. One compound, CJJ300, inhibited TGF-ß signaling by disrupting the formation of the TGF-ß-TßR-I-TßR-II signaling complex. Treatment of A549 cells with CJJ300 resulted in the inhibition of downstream signaling events such as the phosphorylation of key factors along the TGF-ß pathway and the induction of EMT markers. Concomitant with these effects, CJJ300 significantly inhibited cell migration. The present study describes for the first time a designed molecule that can regulate TGF-ß-induced signaling and EMT by interfering with the PPIs required for the formation of the TGF-ß signaling complex. Therefore, CJJ300 can be an important lead compound with which to study TGF-ß signaling and to design more potent TGF-ß signaling antagonists.