Figure 3
RNA-Seq analysis reveals that ONC201 restricts the expression of cell cycle progression and DNA repair pathway genes. (a) Schematic representation of the treatment in which PC3 cells have been primed with ONC201 before being radiated with single doses of Xrad (CorelDRAW Graphics Suite 2019, 64-Bit). (b) Pathway analysis of our genes of interest in KEGG Human 2019 pathway database, after RNA-seq analysis of PC3 cells treated as shown in (a), revealed that the most impacted pathway is cell cycle related (iEnrichr web tool66,67). (c) Heatmaps analysis of RNAseq data (log2 of gene counts) of PC3 cells treated as shown in (a). Cells were harvested at 24 h (c, i and ii top) and 72 h after the irradiation (c, i and ii bottom). The genes listed are the most differentially regulated (see Methods for details on the analysis with RStudio software, Version 1.2.5033, release name "Orange Blossom" (RStudio Team (2020). RStudio: Integrated Development for R. RStudio, PBC, Boston, MA URL http://www.rstudio.com/). (d) Validation through TR-PCR of the five most relevant and differentially regulated genes at 72 h from the final radiation dose (RRM2, CDK2, PLK1, MKI67, CDK1). One-way ANOVA test has been run comparing treated vs Vehicle and No Radiation on n = 3 experiments, **p < 0.01; ***p < 0.001 (± SD). (e) Schematic representation of the cell cycle check points (RRM2, CDK2, PLK1, MIK67, CDK1) were drawn utilizing CorelDRAW Graphics Suite (CorelDRAW Graphics Suite 2019, 64-Bit).
