20 mm glass bottom cell culture dish

Cells were seeded
in 6-well plates or a 20 mm Glass Bottom Cell Culture Dish (NEST,
China) overnight and then treated with irradiation or drugs. The cells
were incubated for 48 h, 72 h, or other time, and the C11 BODIPY 581/591
assay kit (GLPBIO, USA, GC40165) was used following the manufacturer’s
manual. The ROS Assay Kit (GOYOO, China, GY044) was used following
the manufacturer’s manual. Lipid peroxidation levels and ROS
levels were detected by flow cytometry through a BD Accuri C6 Plus
(BD, USA).

Flow cytometry analysis was performed to study the cellular uptake of the nanoparticles. Mz-ChA-1 cells (5 × 10⁴) were seeded in 24-well plates. After 24 h growth, the cells were incubated for 4 h with nanoparticles prepared with AF647-PCX and FAM-anti-miRNA (w/w =2, 100 nM FAM-anti-miRNA). The cells were then trypsinized, washed with cold PBS, and subjected to analysis using a BD FACS Calibur flow cytometer (BD Bioscience, Bedford, MA). The results were processed using FlowJo software. Intracellular localization of AF647-PCX/FAM-anti-miRNA was also observed by confocal laser scanning microscopy. Cells were cultured on a 20 mm glass-bottom cell culture dish (Nest) using 1 × 10⁵ cells per dish. After 24 h, the medium was replaced with fresh medium and AF647-PCX/FAM-anti-miRNA nanoparticles were added (100 nM FAM-anti-miRNA). After incubation for 4 h, the cells were washed twice with PBS, stained with Hoechst 33258 for 10 min. The cells were visualized using a LSM 800 Laser Scanning Microscope (Zeiss, Jena, Germany). Moreover, to follow the endosomal release of nanoparticles, AF647-PCX/anti-miRNA nanoparticles were incubated with cells for 4 h. Then, the cells were washed twice with PBS and stained with LysoTracker Red DND-99 (Life Technology, USA) for 30 min. Finally, the cells were rinsed three times with PBS and visualized with a confocal microscope.