Fig. 1.

Irradiated hTERT-RPE-1 cells exhibit typical markers of cellular senescence accompanied by increased reactive oxygen species (ROS) levels and oxidative stress response. (A) Cells were irradiated by a dose of 20 Gy, and the senescence-associated beta-galactosidase activity (left panel), EdU incorporation (middle panel), and DNA damage (right panel) 8 days after irradiation were compared to proliferating control cells. (B) Elevated protein level of the cell cycle inhibitor p21 in irradiated and proliferating control cells. β-tubulin (TUBB) was used as a loading control. (C) Increased phosphorylation of p38MAP kinase (p38) and its protein level in irradiated and control cells. β-tubulin (TUBB) was used as a loading control. (D) ROS levels were evaluated 8 days after irradiation by flow cytometry (MitoSOX™ Red Mitochondrial Superoxide Indicator, CellROX™ Deep Red, and BODIPY™ C11, see Methods) to quantify intracellular ROS and Amplex™ Red Hydrogen Peroxide/Peroxidase Assay to quantify extracellular H₂O₂ production. Fold changes relative to proliferating control cells are shown and were evaluated using one-sample t-test. The bar graphs show mean ± standard deviation from at least 3 biological replicates. (E) Volume estimation of irradiated and proliferating control cells; the scatter dot plots summarize all measurements from three independent replicates, and the black lines indicate mean ± standard deviation. P-value was estimated using Mann-Whitney test. (F) The lipid membrane compartment content was estimated using the BODIPY™ C11 incorporation. Fold changes between senescent and control cells are shown; the black lines indicate mean ± standard deviation, and the p-value was estimated using one-sample t-test (compared to 1). (D-F) The asterisks indicate statistical significance; *p < 0.05, **p < 0.01, ***p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)