Development, Optimization, and Validation of a TGF-β1-induced Common Mediator Smad4 Nuclear Translocation High...

We describe the development, optimization and validation of a high content imaging assay to quantify the TGF-β1-induced nuclear translocation of common mediator Smad4 (co-Smad4) in the A549 human lung epithelial carcinoma cell line. The 384-well assay was optimized with respect to A549 cell seeding density, serum starvation, TGF-β1 concentration and time of exposure, DMSO tolerance, sample preparation procedures, primary and secondary antibody dilutions, image acquisition settings together with image segmentation and analysis parameters. TGF-β1 treatment of A549 cells dramatically altered the distribution of co-Smad4 antibody staining to a predominantly nuclear co-localization phenotype and image segmentation and analysis using the translocation enhanced module provided a robust assay signal window. TGF-β1 treatment increased the FITC channel 2 co-Smad4 Nuclear to Cytoplasm average intensity ratio from 1 to 5 quantifying the robust co-Smad 4 translocation response from the cytoplasm into the nucleus. The TGF-β1-induced co-Smad4 nuclear translocation assay enabled relative potency comparisons of TGF-β signaling inhibitors with distinct mechanisms of action: TGF-β receptor TGFBR1 and TGFBR2 kinase inhibitors LY364947 and GW788388, TGF-β neutralizing antibody 1D11, and TGF-β receptor complex assembly inhibitors mmTGF-β2-7M and mmTGF-β2-7M-2R. The co-Smad4 nuclear translocation assay was both robust and reproducible and could be implemented as a primary HTS assay to identify inhibitors of TGF-β signaling pathway activation.