Multimode reader

Cells (0.5–1 × 10⁴) were seeded in 96-well plates and exposed to PFK158 (0–30 μM) for 24 and 48 h, and the inhibitory concentrations 50% (IC₅₀) values were determined by MTT assays as previously described^{25 (link)}. Briefly, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT, 100 μg per well) was added to fresh culture media after required drug incubation and further incubated for 3 h at 37 °C. Generated formazan crystals were dissolved in DMSO and optical density was measured at 550 nm in a multimode reader (Biotek, USA). Control cells were exposed to the highest volume of the vehicle.

Mitochondrial membrane potential was determined using Mitochondrial Membrane Potential Assay Kit with JC-1 (Beyotime, #C2006, China). Purified mitochondria were mixed with JC-1 buffer (1×). Fluorescence intensity was detected with Multi-Mode Reader (BioTek) at an excitation of 485 nm and emission wavelength of 535 nm for JC-1 monomers and an excitation of 535 nm and emission wavelength of 595 nm for J-aggregates. The mitochondrial membrane potential was presented as the ratio of J-aggregates to monomers.

Prior to the assay, PC3 and DU145 were grown to 80% confluency and starved in serum free media for 24 h. Cell invasion assay was performed using Trevigen Cultrex® 96 insert cell invasion/ migration chamber kit from Sigma Aldrich as per manufacturer protocol. Briefly, the upper chambers of the 96-well plate were coated with 50 µL 0.6 × BME solution and the plate was incubated at 37 °C overnight. Next day, cells were harvested with trypsin/EDTA and centrifuged for 10 min at 1400 rpm. The supernatant was removed and cells were washed with PBS and resuspended in serum-free media to an approximately 1 × 10⁶ cells/mL. The plate was carefully aspirated, and 50 µL of the cell suspension was added to each well (upper chamber). To the bottom chamber of each well, 150 µL of media supplemented with 10% FBS was added. Finally, the two compounds (at concentration IC₂₀) were added to the cell-containing wells. After 48 h incubation, the upper chambers were carefully aspirated, washed with cell wash buffer and 150 µL of Calcein-AM solution was added to each well and incubated for one hour. Fluorescence was measured at 485 nm excitation, 520 nm emission using multi-mode reader (BioTek, Winooski, VT, USA).

The MAC-T cells were collected 48 h after transfection. Total RNA was extracted from the transfected MAC-T using the TriZol kit (Takara Biomedical Technology Co., Ltd., Beijing, China) following the manufacturer’s protocol. The concentration and quality of RNA were determined using agarose gel electrophoresis and a Multi-Mode Reader (BioTek, Winooski, VT, USA) (Table S1). High-quality RNA samples were used to construct cDNA libraries as described below.

Cell proliferation was analyzed using the CCK-8 cell proliferation and cytotoxicity assay kit according to the manufacturer’s instructions (Solarbio Biotechnology Co., Ltd., Beijing, China). Briefly, cells were seeded in a 96-well plate at a density of 5 × 10³ cells/well and cultured with complete DMEM. After 24 h, they were incubated with 0–10⁻⁴ M of VK₂ (Sigma-Aldrich) for 6, 12, 24, and 48 h. CCK-8 solution was added to the 96-well plate and incubated for 1 h. Then, the 96-well plate was read with a multimode reader (BioTek, Winooski, VT, USA) to obtain the absorbance at 450 nm, and to calculate the cell viability.

ATP levels in mitochondria were detected using an ATP Assay Kit (Beyotime, #S0027, China) according to the manufactures' instructions. 100 μL ATP working reagent was added to a 96-well plate and put at room temperature for 5 min and then added with 20 μL of purified mitochondria per-well. The ATP levels were tested using a Multi-mode reader (BioTek) and calculated according to the standard ATP curve.