Uorescence microscope

Cell proliferation was examined using Ye uor 488 EdU Imaging Kit (Yeasen, Shanghai, China). Cells were rst incubated with EdU at 37°C for 4 h.
After xation and permeabilization, the cells were reacted with 1 mL Click-iT reaction cocktail in darkness for 30 min. For nuclear staining, cells were incubated with DAPI. Finally, the uorescence was observed under a uorescence microscope (Nikon, Tokyo, Japan).

Sections were treated with 0.3% Triton and 10% goat serum for 1 h at room temperature, then incubation overnight at 4 °C with the indicated primary antibody. Sections were then incubated with the indicated secondary antibodies at room temperature in PBS containing 10% normal goat serum for 1 h. Slices were mounted onto slides, embedded in Fluoroshield™ with DAPI, and enclosed under a coverslip. Images were acquired using a Nikon uorescence microscope or a confocal microscope equipped with a 63× (N.A. 1.25) glycerol immersion objective.
Western blot analysis 20 µg total protein per lane separated by SDS-PAGE using 12% precast polyacrylamide gels at 120 V for 90 min. Separated proteins were then transferred to polyvinylidene uoride membranes at 100 V for 2 h. Membranes were blocked with 5% BSA at room temperature for 1 h and incubated with the indicated primary antibodies overnight at 4 °C, followed by incubation with anti-rabbit or anti-mouse immunoglobulin G secondary antibody for 1 h.

Sections were permeabilized with 0.3% Triton and blocked with 10% goat serum for 1 h at room temperature, then incubated overnight at 4 °C with the indicated primary antibody. Immunolabeled sections were then incubated with the indicated secondary antibodies at room temperature in PBS containing 10% normal goat serum for 1 h. Slices were mounted onto slides, embedded in Fluoroshield ™ with DAPI, and enclosed under a coverslip. Images were acquired using a Nikon uorescence microscope or a confocal microscope equipped with a 63× (N.A. 1.25) glycerol immersion objective.

For the generation of the PC-3 tumor organoids, cells were seeded into 96-well round-bottom ultra-low attachment plates (Corning) at 2000 viable cells per well. The PC-3 spheroids were grown in RPMI medium with 10 % FBS. The plates were incubated for 5 days at 37 °C, during which they formed spheroids. Then, the spheroids were treated with 200 μg/ml HMT for 48 h. For the apoptosis analysis, 2 μM CELL Event (Invitrogen, USA) was added to each well for 1 h. Pictures were obtained using a uorescence microscope (Nikon, Tokyo, Japan). Fluorescence intensity of caspase-3/7 was evaluated using the ImageJ software. For the calculation of uorescence intensity with ImageJ, two images of each well were analyzed and their average intensity was used for the statistical analysis.

A Cell-Light EdU DNA Cell Proliferation Kit (KeyGEN BioTECH, China) was carried out to evaluate cell proliferation. Brie y, cells were grown into 96-well plates. Then, cells were treated with 50 μmol/L EdU for 2 hours. Then, cells were xed using 4 % paraformaldehyde and then 1×Apollo reaction cocktail was used. Cell nuclei was stained using DAPI for 15 min. We captured the images using a uorescence microscope (Nikon, Tokyo, Japan).
Apoptosis assays PI and FITC-labeled annexin V (BD Biosciences, New Jersey, USA) staining was used to assess cell apoptosis. Cells were washed twice using PBS and resuspended using 100 μL 1× binding buffer, and then incubated with 5μL FITC-annexin V and PI with no light. Then, 400 μL 1× binding buffer was used. Within 1 hour, cells were exposed to ow cytometry analysis.

For Oil Red O staining, cells were xed in a 4% paraformaldehyde solution for 30 min, induced with 60% Oil Red O for 30 min, and washed three times with PBS, and then the cells were visualized by phasecontrast microscope (IS-Elements software, Nikon ECLIPSE, Tokyo, Japan). Oil Red O was extracted with 100% isopropanol, and its relative concentration was determined by measuring the absorbance at 510 nm. After being xed in 4% paraformaldehyde solution for 15 min, cells were stained with Bodipy (1 μg/mL; Life Technologies, Carlsbad, CA, USA) or AdipoRed (30 μl / ml; Lonza, USA) for 20 min; the sections were washed with PBS three times for 5 min each. For nuclear visualization, DAPI (4′,6diamidino-2-phenylindole; Roche) was incubated for 10 min, then the section was rinsed with PBS. After treatment, the sections were observed under uorescence microscope (Nikon, Tokyo, Japan).