Figure 2.

Combined treatment with radiation and ATR inhibitors increases ATP secretion. (A) Experimental set-up for measurement of ATP secretion, exemplified for the 72 hour time-point. Treated samples were incubated for 72 hours. Six hours prior to medium harvest, the medium was replaced with a reduced amount of serum-free medium. The media were collected from the samples, and centrifuged twice for cell exclusion, before the medium supernatants were analyzed by use of the CellTiter-Glo assay. Same set-up was employed for the other time-points, still with medium replacement for the last six hours. (B-D) Results from experiments performed as in (A) for U2OS (B), H1975 (C) and SW900 cells (D) at indicated time-points after treatment with ionizing radiation (5 Gy; IR) and/or ATR inhibitors (VE822 or AZD6738 at indicated concentration). Bar charts show crude relative luminescence values (relative luminescence units; RLU), which is proportional to the extracellular concentration of ATP. p values were calculated for difference between co-treatments and irradiation alone or ATR inhibition alone. For statistical analysis between groups of different size, only data that paired up from the same experiments were included. (E) Results from experiments performed as in (A) in H460 cells, presented as for (B-D) (left). To correct for vast differences in cell count after treatment in this cell line, the relative luminescence values were normalized to cell counts at time of harvest (right). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.