br Colony formation and cell cycle
2.3. Colony formation and 30931-67-0 assay
Cells plated at low density (100 cells/well) in 6-well plate were cultured in their medium complemented or not, with 10, 25 μmol/L Bou. After 10 days of culture, colonies were fixed and stained with 0.05% (w/v) crystal violet in 70% (v/v) ethanol for 2 h. The stained cells were dissolved in 1% SDS and the corresponding optical density (OD) was read at 570 nm. For cell cycle determination, cells were col-lected and washed twice with phosphate buﬀered solution (PBS), then fixed with 70% ethanol in PBS at −20 °C overnight. After being washed with PBS, cells were then stained with 10 μg/ml PI dye for 30 min in the dark. Finally, cells were washed and analyzed using Influx TM flow cytometer. Data were analysis by Expo 32 Software.
2.4. Glucose uptake assay
Glucose uptake was monitored using fluorescent 2-NBDG and quantification Kit. After treatment, cells were washed with PBS and co-incubated with 30 μM 2-NBDG for 30 min in the dark at 37 °C, respec-tively. Microscopies were captured by Zeiss Confocal microscopy under the excitation of 488 nm.
2.5. OCR and ECAR determination
HCT-116 cells were seeded in XF 96-well cell culture microplate in quintuplicate at 5000 cells/well in 0.1 ml growth medium and then incubated at 37 °C in 5% CO2. Each group was carried out in 8 re-plicates. Mitochondria isolated from tumors of mice were planted into XF 96-well cell culture and centrifuged at 2000 g for 5 min. Assays were initiated by removing the growth medium from each well and replacing it with 100 μl of assay medium pre-warmed to 37 °C. The cells were incubated at 37 °C for 30 min to allow media temperature and pH to reach equilibrium before the first rate measurement. Prior to each rate measurement, the XF-96 Analyzer gently mixed the assay media in each well for 10 min to allow the oxygen partial pressure to reach equili-brium. Following mixing, OCR and ECAR were measured simulta-neously for 3–5 min to establish a baseline rate. The assay medium was then gently mixed again for 3–5 min between each rate measurement to restore normal oxygen tension and pH in the microenvironment sur-rounding the cells. After the baseline measurement, 5–20 μl of a testing agent prepared in assay medium was then injected into each well to reach the desired final working concentration. This was followed by mixing for 5–10 min to expedite compound exposure to cellular pro-teins, after which OCR and ECAR measurements were then made. Generally, two to three baseline rates and two or more response rates (after compound addition) were measured, and the average of two baseline rates or test rates was used for data analysis. For time-resolved experiments, multiple measurements as well as compound injections were made at the time points indicated. The values of OCR and ECAR reflect both the metabolic activities of the cells and the number of cells being measured. Typically, at the end of each assay cells are treated for protein extraction.
2.6. Mitochondrial content and oxidation complexes activity determination
For the determination of mitochondria content, the DNA level of cytochrome C (for mitochondria DNA) and 18s rRNA (for nDNA) were measured using PCR assay and quantified by calculating the ratio of mtDNA/nDNA (Rao et al., 2017). Mitochondria in the cultured cells or the tumor tissues collected from animal studies were isolated using
mitochondria isolation kit. For determination of the activity mi-tochondrial complexes, the cells and tumor tissues were washed with ice-cold PBS and lysated with 0.9% NaCL under freeze-thawing cycles. After a centrifugation at the speed of 12000 g, 30 min at 4 °C, the su-pernatant was separated and applied for CS, complex I, II activity and protein levels were measured using a quantification kit (Abcam, China) according to the instructions of manufacturer.
2.7. Electron microscoping
Cells were fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buﬀer (pH 7.4) at 4 °C overnight and post-fixed with 1% osmium tetr-oxide/1% potassium ferrocyanide for 1 h at room temperature. After fixation, cells were stained en bloc with 5% aqueous uranyl acetate overnight at room temperature, dehydrated and embedded in Taab epoxy resin. Ultrathin sections were stained with lead citrate and re-corded using a Megaview 3 digital camera and iTEM software in a Jeol 100-CXII electron microscope.
2.8. Reactive oxygen species detection
Intracellular level of reactive oxygen species was measured using reactive oxygen species probe DCFH-DA. Briefly, cells were exposed to Bou (25 μM) treatment for 24 h. After treatment, cells were incubated with 1 μM DCFH-DA probe in culture medium solution for 30 min in the dark at 37 °C. The microscopy was captured under the excitation of 488 nm. The fluorescence of reactive oxygen species was measured with