Hoechst 33342

Cells seeded on a 24-well glass bottom plate were subjected to the same stimulation conditions as those of the Western blot sample. The collected cells were set immediately on a metal block positioned on ice and were washed five times with ice-cold PBS. Then, 4% PFA was added to cells to fix them at room temperature (RT) for 20 min. The cells were then washed with PBS three times and were incubated with 0.2% Triton X-100 in PBS(+) for 15 min at RT. After cells were washed with PBS(+) supplemented with 0.1% Tween-20 (PBST), they were blocked with gelatin from cold-water fish for 1 h at RT. Then, cells were washed twice with PBST and were stained with Hoechst 33342 (1:1000, #62249; Thermo Fisher Scientific Inc., Waltham, MA, USA) for 10 min at RT. After rabbit anti-Akt antibody was dissolved in 2% BSA (in PBST) with 1:200 dilution, it was incubated at RT for 1 h. After washing with PBST another three times, secondary antibodies dissolved in 2% BSA (in PBST) were added to cells for shaking at RT for 1 h. After the final wash with PBST three times (5 min interval), cells were put to immediate microscopic observation using a confocal microscope (FV1200; Olympus Optical Co. Ltd., Tokyo, Japan) with a 10× or 60× lens and imaging acquisition condition: 10 μs pixel⁻¹, 568 nm laser excitation, and 405 nm laser for Hoechst 33342, excitation.

After an incubation period of 4 days, the spheroids were treated with 0.1 mg/mL of the indicated compounds for 72 h. The spheroids were washed twice with DPBS and CPT-loaded aggregates were stained with PI for 10 min to visualize dead cells. EPI-loaded aggregates were counterstained with Hoechst 33,342 (Fluka, Buchs, Switzerland). Fluorescence imaging was performed by using an Olympus IX73 inverted microscope with DAPI channel for Hoechst 33,342 and the CPT-loaded aggregates (λ_excitation = 345 nm, λ_emission = 455 nm) and Cy3 channel for PI and the EPI-loaded aggregates (λ_excitation = 550 nm, λ_emission = 565 nm).