2.5. Acridine Orange Staining and GFP Transfection

For acridine orange staining, DU145 and PC-3 cells were seeded at 15 × 10⁴ density in coverslips placed in a six-well plate and incubated for 24 h at 37 °C in a CO₂ incubator. The cells were treated with JNK inhibitor for 48 h. After 48 h, the cells were washed with PBS and stained with 1 µl/mL 3,6-Acridine diamine orange (AO) (5mg/mL stock concentration in DMSO); this was performed for 10 min in the 37 °C incubator. Stained acidic vacuoles (lysosomes, endosomes, and autophagosomes) were determined by fluorescence microscopy in excitation (460 nm) and emission (650 nm).

For GFP–LC3 transfection, DU145 and PC-3 PCa cells were seeded at 1 × 10⁵ density in coverslips placed in a six-well plate. The GFP–LC3 plasmid was transfected into cells with FuGene Transfection Reagent (Promega, Southampton, United Kingdom) in a 3:1 ratio. One µl of plasmid and 3 µl of transfection reagent were placed into serum-free media in two different centrifuge tubes and incubated for 10 min. Then the two tubes were combined and gently pipetted. After pipetting, they were incubated for 20 min. Lysates were added drop wise to the serum media on top of the cells. The cells were incubated for 48 h at 37 °C in a CO₂ incubator for transfection. GFP–LC3 transfected cells were treated with JNKi (10 µM) at 48 h, and then examined by fluorescence microscopy.