Fig. 4. Evaluation for hallmarks of ICD in A375 MCTS. Evaluation for hallmarks of ICD in A375 MCTS upon treatment with Ir-pbt-Bpa (5 μM) in the dark or upon two-photon irradiation (750 nm, 50 mW, 5 min). (a) One-(OPM) and two-photon (TPM) excited z-stack confocal laser scanning microscopy upon incubation for 12 h. (b) Release of nuclear high-mobility group box 1 protein into the cell culture supernatant. (c) Release of adenosine triphosphate into the cell culture supernatant. (d) Immunofluorescence confocal laser scanning microscopy stained with a nuclear high-mobility group box 1 protein-specific antibody (red) and 4′,6-diamidino-2-phenylindol (blue). Scale bar: 100 μm.