Intracellular ferrous ion distribution was assessed using a FeRhoNox-1 staining kit (Goryo Chemical, Inc.). PC12 cells (5×104/well) were seeded in a 24-well microplate overnight. Following transfection with miR-335 mimic, miR-335 inhibitor, miR-335 NC, or siRNA-FTH1, and stimulation with 6-OHDA (50 µM) for 24 h, the cell culture medium was removed. The cells were then incubated with 1 µM FeRhoNox-1 at 37°C for 1 h. After washing with PBS 3 times, Fe2+ distribution was imaged using a Cytation 5™ Cell Imaging Multi-Mode Reader (BioTek Instruments, Inc.).

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