Both PC-3 and LNCaP cells were cultured and treated with zinc as required, and immunofluorescence staining was performed. Briefly, after being washed with PBS, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature. To identify TR3 protein expression, cells were first incubated with an anti-TR3 immunoglobin G (IgG) antibody (sc365113; Santa Cruz Biotechnology Inc., Dallas, TX, USA) and then with a corresponding FITC-conjugated anti-IgG antibody (AS10115; Agrisera, Vasterbotten, Sweden) as the secondary antibody. To visualize nuclei, cells were stained with 50 µg/mL of 4,6-diamidino-2-phenylindole (B28718; Sigma-Aldrich) containing 100 mg/mL of Dnase-free Rnase A. To visualize mitochondria, cells were incubated with an anti-HSP60 mouse IgG antibody (sc136291; Santa Cruz Biotechnology, Inc.) and reacted with a Texas red-conjugated anti–mouse IgG (sc69786; Santa Cruz Biotechnology, Inc.) as the secondary antibody.
Additionally, to display the translocation of cytochrome c in PC-3 and LNCaP cells, the experimental procedure was performed as described by the manufacturer’s protocol. Cells were incubated with an anti-cytochrome c antibody (sc13156; Santa Cruz Biotechnology, Inc.) and then the FITC-conjugated antibody. Immunofluorescence staining procedures for both nuclei and mitochondria were then conducted.
Additionally, to display the translocation of cytochrome c in PC-3 and LNCaP cells, the experimental procedure was performed as described by the manufacturer’s protocol. Cells were incubated with an anti-cytochrome c antibody (sc13156; Santa Cruz Biotechnology, Inc.) and then the FITC-conjugated antibody. Immunofluorescence staining procedures for both nuclei and mitochondria were then conducted.