Figure 3. Gemcitabine prior to radiation (XRT) is more efficacious than gemcitabine (GEM) alone or XRT prior to GEM due to differential modulation of HuR and dCK proteins. (A) Anchorage-independent colony formation assays were performed for quantification of colonies formation in MiaPaCa2 cells treated with gemcitabine (1 uM) alone or with gemcitabine preceded or followed by radiation (2 Gy). (B) PicoGreen cell survival assay on cells treated with radiation or with gemcitabine preceded or followed by radiation. (C) PicoGreen cell survival assay after HuR silencing with siRNA oligos. (D) RT-qPCR validation of HuR siRNA treatment in MiaPaCa2 cells. (E) Western blot for dCK, HuR, and α-tubulin (loading control) on lysates obtained from cells treated with gemcitabine alone, radiation alone, gemcitabine prior to radiation and radiation prior to gemcitabine.