Imaging cell chambers

Live imaging of the cells treated with compound TAL3PYR was performed on the A549 cell line. Cells were seeded in Ibidi imaging cell chambers (Ibidi^®, Gräfelfing, Germany) in 500 μL of medium, 5 × 10⁴ cells/well, and wer eleft in the cell incubator for 48 h (37 °C, 5% CO₂). Subsequently, cells were treated with a 10 μM solution of the respective compound and left in the cell incubator for 90 min to allow the compound to enter the cells. After incubation, the medium was replaced with 500 μL of fresh medium. The influence of the compounds on A549 cells before and after photoactivation (λ_exc = 405 nm, λ_em = 450–550 nm) was visualised using a Leica SP8 X confocal microscope (Leica Microsystems, Wetzlar, Germany).

Live imaging of the cells treated with compounds was performed on the HeLa cell line. Cells were seeded in Ibidi imaging cell chambers (Ibidi^®, Gräfelfing, Germany) in 500 μL of a medium, with the concentration of 7.5 × 10⁴ cells/well and left in the cell incubator for 48 h (37 °C, 5% CO₂). After two days, the cells were treated with a 1 μM solution of each compound and left in the cell incubator for 60 min to allow the compound to enter the cells. After the incubation, the medium was changed, and 500 μL of 100 nM MitoTracker Deep Red solution (Invitrogen, Molecular Probes) was added to the chambers. Cells were incubated for 20 min (37 °C, 5% CO₂), allowing MitoTracker to enter the cells. After incubation, the medium was replaced with 500 μL of fresh medium. Co-localization of compounds (λ_exc = 405 nm, λ_em = 470–670 nm) and mitochondria (MitoTracker λ_exc = 644 nm, λ_em = 665 nm) was then visualized and confirmed using Leica SP8 X confocal microscope (Leica Microsystems, Wetzlar, Germany). Control and untreated cells excluded the presence of foreign fluorescence.