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. 2021 Jun 8;22(8):e52032. doi: 10.15252/embr.202052032

Figure 3. Mechanisms triggering senescence contribute to NK cell recognition in ArCK cells.

Figure 3

  1. γ‐H2AX foci were analyzed in ArCK‐ and G1‐arrested cells (generated as described in Fig 2). At least 50 cells were analyzed per condition per replicate. n = 2 biological replicates; individual values and mean are shown. The distribution of the foci number in each treated condition was compared to that of control using Kolmogorov–Smirnov test. Ctrl vs. Doxo, P < 0.0001; Ctrl vs. Palbo, P = 0.07, n.s.; Ctrl vs. Nutlin, P = 0.11, n.s.; Ctrl vs. ArCK, P = 0.0007.
  2. p53 and p21 levels were determined by Western blot analysis. Vinculin was used as loading control. Results were comparable between 2 biological replicates.
  3. The degree of senescence was measured by senescence‐associated β‐galactosidase (β‐gal) activity. The graph shows the percentage of β‐gal‐positive cells. At least 100 cells were analyzed per condition per replicate. n = 3 biological replicates; mean ± SEM. Ctrl vs. Doxo, ***P = 0.0006; Ctrl vs. Palbo, *P = 0.025; Ctrl vs. Nutlin, *P = 0.016; Ctrl vs. Torin, P = 0.806, n.s.; Ctrl vs. ArCK, ***P = 0.0001; unpaired t‐test.
  4. Secreted cytokine and interferon levels were determined in cell supernatants. Media were collected after 36 h of incubation with cells grown as described in Figs 1 and 2. Cytokine and interferon levels were shown as fold change normalized to euploid control cells. Individual values and mean are shown; n = 2 biological replicates.
  5. NK cell medium was incubated with either euploid control or ArCK cells for 12 h. At the time of NK cell addition, media were switched between ArCK and euploid control cells (Ctrl). NK cell killing was measured as described in Fig 1C. For reference, NK cell killing of ArCK and euploid control cells (Ctrl) without medium switch were performed side by side and plotted on the graph. Black, euploid control cells without medium switch; red, ArCK cells without medium switch; blue, euploid control cells in ArCK cell condition medium; green, ArCK cells in euploid control cell condition medium. n = 3 biological replicates; mean ± SEM. Ctrl vs. ArCK, P < 0.0001; Ctrl vs. Ctrl in ArCK med, P < 0.0001; Ctrl vs. ArCK in Ctrl med, P < 0.0001; ArCK vs. ArCK in Ctrl med, P = 0.0002; KS test.