Hochest 33258

For Hochest 33258 staining, progress was achieved by following the manufacturer’s instructions. Briefly, the cells that were grown on Bioflex plates were incubated with fixation buffer (0.5 ml per well) for 10 min at room temperature and washed twice with PBS. Then, the cells were stained with 1 ug/mL Hochest 33258(Invitrogen, H3569) for 10 min in the dark, washed twice with PBS, and examined by fluorescence microscopy. The Hoechst reagent was taken up by the nuclei of the cells, and apoptotic cells exhibited bright blue fluorescent, condensed, and fragmented nuclei. The apoptotic index (AI) was calculated as the percentage of apoptotic nuclei per total nuclei number per field as ref [29 (link)].

LNCaP cells were plated on poly-L-lysine-coated coverslips (Marienfeld, 0111530) and after being treated as indicated, they were fixed with 4% paraformaldehyde in 1× PBS for 15 minutes. Next the coverslips were mounted in mounting solution [20 mM n-propylgallate, 80% Glycerol, 20% 1×PBS], and visualized by phase-contrast microscopy (Lecia, DMI4000B). The length of the neurites on the coverslips was analyzed by MetaMorph (Molecular Devices, Neurite Outgrowth). LNCaP cells show low basic level of NED under normal culture condition (RPMI 1640 supplemented with 10% FBS). The average neurite length of control cells cultured in RPMI 1640 containing 10% FBS in each experiments was used as 1, and a comparison was then made for neurite length obtained after IL-6 treatments. The LNCaP-eGFP-LC3 cells were prepared as described above for LNCaP cells except for staining with Hochest 33258 (Invitrogen, H3569) before mounting. The eGFP-LC3 images were visualized using a Lecia DMI4000B fluorescence microscope with a 63× lens and were analyzed by MetaMorph (Molecular Devices, Transflour). LNCaP cells show low basic level of autophagy under normal culture condition. To distinguish the autophagy induction, cells displaying more than 50 intense eGFP-LC3 aggregates of 5 to 7.5 µm and 15 intense eGFP-LC3 aggregates of 7.5 to 20 µm were counted as autophagy-positive cells after induction.