Jang et al. 10.1073/pnas.0706662104. |
Fig. 6. TrkA agonists screening. (A) The schematic flowchart of TrkA agonists' screening strategy. (B) The representative microscopic pictures from SN56 and T17 cells pretreated with NGF or gambogic amide, followed by staurosporine (STS). The apoptotic cells were stained with red fluorescent dye activated by caspase-3.
SI Text
A Cell-Based Screen for TrkA-Expressing Cells.
We cultured T17 cells in 96-well plates and preincubated the cells with 10 mM compounds for 30 min, followed by 1 mM staurosporine (STS) treatment for 9 h. MR(DEVD)2 was introduced into the cells 1 h before examination under a fluorescence microscope. The apoptotic cells were red, whereas live cells had no signal. As a positive control, NGF substantially decreased the red cell numbers compared to DMSO control. Using the caspase-3-activated fluorescent dye as a visual assay, we screened 2,000 biologically active compounds from the Spectrum Collection Library. Thirty-one compounds selectively protected T17, but not SN56, cells from STS-initiated apoptosis, indicating that these compounds might act either directly through TrkA receptor or through its downstream signaling effectors. The representative results from the screening are shown (Fig. 6B Left). Even in the absence of NGF, T17 exhibited a stronger antiapoptotic effect than its parental SN56 cells, indicating that the overexpression of TrkA suppresses caspase-3 activation. NGF treatment further enhanced this effect (SI Fig. 6B Right). These observations were verified by immunoblotting with active caspase-3 antibody. The positive compounds were further validated by an independent cell viability assay (data not shown).