Figure 5. Combination treatment of GNF5 and docetaxel induces cytoplasmic localization of Yap1 and decreases expression of downstream transcription targets of Yap1 compared to vehicle control treated lung adenocarcinomas.
GFP+ cells were isolated from KRASG12D/+; p53−/−; Rosa26-fGFP mice two weeks after treatment with vehicle, docetaxel, GNF5, or combination treatment and 12 weeks after induction of tumors with adenovirus. A. Immunofluorescence staining for Yap1 shows a decrease in Yap1 nuclear localization in mice treated with GNF5 and docetaxel compared to vehicle control treated mice. Scale = 75 μm. B. Higher magnification images are provided to show sub-cellular localization of Yap1 in control and combination treatment mice. Scale = 7.5 μm. C. Western blot analysis showed no significant difference in total Yap1 protein expression in double treated mice compared to vehicle treated control mice. Phospho-CrkL expression is a marker of ABL kinase activity. D. RT-qPCR analysis showed a decrease in mRNA transcript expression of the downstream Yap1 target, Birc5, which encodes the protein survivin, in mice treated with both GNF5 and docetaxel compared to vehicle control treated mice or mice treated with GNF5 or docetaxel alone (n = 3-4 mice per group, each RT-qPCR assay performed in triplicate). E. Western blot analysis showed a decrease in protein expression of downstream transcriptional targets of Yap1, including survivin, c-Myc, S100A4, and cyclin D1. Graphs depict means and S.E.M.