Fig. 2. Cellular localization and photocytotoxicity. (a) Confocal laser scanning microscopy images of A375 cells upon incubation with Ir-pbt-Bpa (2 μM) for 4 h and MitoTracker™ Red (Mito-R) for 0.5 h. Scale bar: 10 μm. (b) Subcellular distribution of Ir-pbt-Bpa (2 μM) upon incubation in A375 cells for 4 h determined by inductively coupled plasma mass spectrometry. Flow cytometry spectrum upon treatment of A375 cells with Ir-pbt-Bpa to investigate the ability of the compound to (c) generate ROS, (d) produce lipid peroxides or (e) release Ca²⁺ ions. (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 (concentration: IC₅₀ value) for 4 h in the dark, (IV) cells were incubated with Ir-pbt-Bpa (concentration: 2× IC₅₀ value) for 4 h in the dark; (V) cells were incubated with Ir-pbt-Bpa (concentration: IC₅₀ value) for 4 h and exposed to irradiation (405 nm LED, 0.75 J cm⁻²), (IV) cells were incubated with Ir-pbt-Bpa (concentration: 2× IC₅₀ value) for 4 h and exposed to irradiation (405 nm LED, 0.75 J cm⁻²). The dashed lines represent the negative controls without dye. (f) Fluorescence microscopy images of A375 cells upon incubation with Ir-pbt-Bpa for 4 h and calcein-AM (live, green)/propidium iodide (dead, red) in the dark or upon irradiation (405 nm LED, 0.75 J cm⁻²). Scale bar: 100 μm. (g) Image of the western blot analysis of the treatment of A375 cells with Ir-pbt-Bpa. (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), (V) cells were incubated with Ir-pbt-Bpa (2 μM) for 4 h in the dark; (IV) cells were incubated with Ir-pbt-Bpa (2 μM) for 4 h and exposed to irradiation (405 nm LED, 0.75 J cm⁻²).