Figure 4.

The transfer of cell-conditioned medium from irradiated cells to nonirradiated cells reduced the clonogenic survival of tumor cells (RIBE) and was accompanied by the expression of cytokines mediated by activation of DNA sensing pathways. (a) Clonogenic survival of B16F10 tumor cells after the transfer of cell-conditioned medium of control nonirradiated B16F10 cells (Ctrl group) or 6 Gy irradiated B16F10 cells (IR group); n = 4. (b) Clonogenic survival of B16F10 tumor cells after the transfer of cell-conditioned medium of control nonirradiated RAW 264.7 macrophages (Ctrl group) or 6 Gy irradiated RAW 264.7 macrophages (IR group); n = 3. (c) The protein expression fold change of TNFα and IFNβ at 24 h (24 h group) and 48 h (48 h group) after irradiation of B16F10 cells normalized to control nonirradiated cells (Ctrl). The expression was evaluated by intracellular flow cytometry and represents the frequency of parent population (TNFα or IFNβ positive subset of single live cells); n = 4–6. (d) The protein expression fold change of IL1β and IFNβ at 24 h (24 h group) and 48 h (48 h group) after irradiation of RAW 264.7 macrophages normalized to control nonirradiated cells (Ctrl). The expression was evaluated by intracellular flow cytometry and represents the frequency of parent population (IL1β or IFNβ positive subset of single live cells); n = 5–6. (e) Relative expression of cytokine receptors Tnfr, Ifnar1, Ifnar2, Il1r1, and Il1r2 in B16F10 cells normalized to Gapdh expression demonstrating present binding targets for released cytokines; n = 6. Statistical significance was determined by one-way ANOVA followed by a Dunnett’s multiple comparisons test for panels A, B, C, D or Tukey’s multiple comparisons test for panel E, n = number of biological replicates. * p < 0.05, ** p < 0.01, **** p < 0.0001 vs. Ctrl. n = number of biological replicates.