Fig. 3. Evaluation for hallmarks of ICD in A375 cells. Evaluation for hallmarks of ICD in A375 cells upon treatment with Ir-Bpa (1 μM) and Ir-pbt-Bpa (1 μM) in the dark or upon irradiation (405 nm LED, 0.75 J cm⁻²). (a) Immunofluorescence confocal laser scanning microscopy stained with a calreticulin-specific antibody. Scale bar: 10 μm. (b) Flow cytometry spectrum to investigate the translocation of calreticulin. (I) cells were kept in the dark; (II) cells were exposed to irradiation (405 nm LED, 0.75 J cm⁻²); (III) cells were incubated with Ir-pbt-Bpa (1 μM) for 4 h in the dark, (IV) cells were incubated with Ir-pbt-Bpa (1 μM) for 4 h and exposed to irradiation (405 nm LED, 0.75 J cm⁻²). (c) Immunofluorescence confocal laser scanning microscopy stained with a nuclear high-mobility group box 1 protein-specific antibody. Scale bar: 10 μm. (d) Release of nuclear high-mobility group box 1 protein into the cell culture supernatant. (e) Release of adenosine triphosphate into the cell culture supernatant.