Co-staining of KCa 3.1 Channels in NSCLC Cells with a Small-Molecule Fluorescent Probe and Antibody-Based Indirect Immunofluorescence.

The Ca2+ activated potassium channel 3.1 (KCa 3.1) is involved in critical steps of the metastatic cascade, such as proliferation, migration, invasion and extravasation. Therefore, a fast and efficient protocol for imaging of KCa 3.1 channels was envisaged. The novel fluorescently labeled small molecule imaging probes 1 and 2 were synthesized by connecting a dimethylpyrrole-based BODIPY dye with a derivative of the KCa 3.1 channel inhibitor senicapoc via linkers of different length. Patch-clamp experiments revealed the inhibition of KCa 3.1 channels by the probes confirming interaction with the channel. Both probes 1 and 2 were able to stain KCa 3.1 channels in non-small-cell lung cancer (NSCLC) cells following a simple, fast and efficient protocol. Pre-incubation with unlabeled senicapoc removed the punctate staining pattern showing the specificity of the new probes 1 and 2. Staining of the channel with the fluorescently labeled senicapoc derivatives 1 or 2 or with antibody-based indirect immunofluorescence yielded identical or very similar densities of stained KCa 3.1 channels. However, co-staining using both methods did not lead to the expected overlapping punctate staining pattern. This observation was explained by docking studies showing that the antibody used for indirect immunofluorescence and the probes 1 and 2 label different channel populations. Whereas the antibody binds at the closed channel conformation, the probes 1 and 2 bind within the open channel.

Role of TGF-ß Signaling in Coupling Bone Remodeling.

TGF-ß signaling plays a key role in the temporal and spatial regulation of bone remodeling. During osteoclast bone resorption, TGF-ß is released from the bone matrix and activated. Active TGF-ß recruits mesenchymal stem cells to the bone resorption pit through the SMAD signaling pathway. Mesenchymal stem cells undergo osteoblast differentiation and deposit new bone filling in the resorption pit and maintaining the structural integrity of the skeleton. Thus, TGF-ß signaling plays a key role in the coupling process and disruptions to the TGF-ß signaling pathway lead to loss of skeletal integrity. This chapter describes methods on how to quantitate bone matrix TGF-ß and assess its role in mesenchymal stem cell migration both in vitro and in vivo.