Acridine orange

For acridine orange experiments, 0.3 × 10⁶ WT or OCA7-KO MNT1 cells were seeded in 35 mm glass bottom dishes with 2 ml 37 °C KGM Gold media and grown for 24 h. To stain cells, media were aspirated from the dish, and cells were incubated with 2 ml acridine orange (Bio-Rad, ICT937) diluted to 1 μM in KGM Gold media for 30 min in a humidified cell culture incubator at 37 °C and 5% CO₂. After the incubation, cells were washed twice for 1 min with 2 ml 37 °C PBS. After washing, 2 ml fresh KGM Gold media were added to the dish, and the cells were placed in the humidified stage incubator of a Zeiss LSM900 microscope and immediately imaged in confocal mode with excitation set to 488 nm and detection of 400 to 700 nm. Images were analyzed by measuring fluorescence intensity of manually drawn ROIs in ImageJ.

Cells were seeded with a density of 62 × 10³ cm², and after reaching confluence, they were stimulated with Ag- and Au-NPs ± PT for 24 h, BafA 50 nM for 2 h or left with medium alone. Acridine orange (Bio-Rad, Hercules, CA, USA) at the concentration of 1 µg/mL was then added to the cells and incubated for 30 min. Cells were washed with PBS, and fluorescence signals (both red and green) were detected with a plate reader (excitation 480 nm; emission 620 nm for red, 550 nm for green). Data are presented as red/green signals ratio as previously described [38 (link),39 (link)]. For the Magic Red assay, HIEC-6 cells were exposed to treatments for 24 h, leupeptin 20 nM for 2 h or left with medium alone. Magic Red (Bio-Rad, Hercules, CA, USA) was then added to the cells and incubated for 30 min. Cells were washed with PBS, and fluorescence signals were detected with a plate reader (excitation 595 nm; emission 635). The unlabeled cells and the noncellular test, run as negative controls, showed null or negligible signals.