Figure 2
Priming Prostate Cancer cells with ONC201 increases the efficacy of irradiation (a) Schematic representation of the treatment in which PC3 cells have been primed with ONC201 before being radiated with single doses of Xrad. (b) Cell counts showed that priming PC3 cells to radiation (Xrad) with ONC201 for 24 h impacts cells survival at 72 h. Radiation Enhancement Ratio (RER) is shown for ONC201 (5–15 µM) and Radiation (2, 4 and 8 Gy) and calculated out of 3 independent experiments. (c) Clonogenic Assay. Priming PC3 cells to radiation (2, 4 and 8 Gy) with ONC201 (0.1–15 µM) for 24 h prevents colony formation for concentrations higher than 1 µM. The cells were treated with ONC201 for 24 h and then either mock irradiated or irradiated with the indicated doses. Post-treatment, the cells were cultured in clonogenic conditions and photographed after 1 week (Images representative of n = 3). (d) ONC201 10 µM has a synergistic effect with all doses of radiation (2, 4 and 8 Gy) at 72 h (CompuSyn). (e) Western Blot analysis of the expression of the main components of the UPR upon priming PC3 cells to radiation (Xrad) with ONC201 for 24 h. Samples were harvested at 72 h from the irradiation (Representative of n = 3). Densitometric analysis of n = 3 WB are shown in Supplementary Fig. 2c. Here the average is expressed as a number embedded within the figure (the SD are within the 5% acceptance of statistical significance).The membranes were cut before the incubation with the primary antibody (see “Materials and methods” for specifications) and either detected alone or together. All the membranes were exposed for 3 min, with the exception of ATF4 and ATF6 (exposed for 6 min), PERK and Tubulin (exposed for 2 min). (f) Cell death analysis through Annaxin V/PI detection of PC3 treated with ONC201 (5 and 10 µM) and Radiation (as described in (a)). Samples were harvested at 72 h. One-way ANOVA test has been run comparing treated vs Vehicle and No Radiation on n = 3 experiments, *p ≤ 0.05; **p < 0.01; ***p < 0.001 (± SD).
