br Cell cycle analysis br
2.3. Cell cycle analysis
After treatment with celastrol (0, 0.25, 0.5, and 1 μM) for 24 h, the 50-23-7 were collected and fixed with 75% ethanol (1 × 106/ml) at -20 °C overnight, washed with phosphate-buﬀered saline (PBS), and treated with RNase I, then stained with propidium iodide (BD Biosciences, San Diego, CA, USA) for 15 min in the dark. Cell cycle analysis was then detected by flow cytometry (BD FACSCanto, BD Biosciences, San Jose, CA, USA).
2.4. Apoptotic analysis
according to the Annexin V-fluorescein isothiocyanate (FITC)/7-AAD kit protocol (BD Biosciences, San Diego, CA, USA). The cells were in-cubated with FITC-conjugated annexin V and 7-AAD for 10 min at room temperature in the dark. Cells were then washed with ice-cold PBS. For each measurement, 1 × 104 cells were acquired and analyzed by flow cytometer after FITC-Annexin V/ 7AAD staining to distinguish and quantify viable cells (annexin V-, 7AAD-), early apoptotic cells (annexin V+, 7AAD-) and late apoptotic cells (annexin+, 7AAD+) (BD FACSCanto, San Jose, CA, USA). Each experiment was performed in triplicate.
2.5. Wound-healing assay
Cells were seeded equally into 6 well plates (5 × 104 cells/well). When the cells grew to 70–80% confluence, an artificial homogenous wound was produced on the monolayer by a sterile 10 μl tip and the detached cell was then removed. The cells were incubated with celas-trol (0, 0.25, 0.5, and 1 μM) for 24 h and the images were taken using an inverted microscope (CKX71, Olympus Corporation, Tokyo, Japan). The wound width was analyzed via Image J software and the migration rate was calculated by the following equation:
% Migration rate = average distance scratch sides (0 h)
− average distance scratch sides (24 h)
/average distance between scratch sides (0 h)
2.6. Transwell invasive assays
Cell invasion assays were studied with 24-well 8 µm pore size Transwell chambers (Corning Inc., Tewksbury, MA, USA). First, 5 × 104 cells in 100 μl of serum-free culture medium containing dif-ferent concentrations of celastrol (0, 0.25, 0.5, and 1 μM) were seeded onto the upper chamber, and the medium containing 10% FBS was placed in the lower chamber. After 24 h, cells in the top chamber were removed using a cotton swab. Cells invading to the bottom chamber were fixed with 4% paraformaldehyde and then stained with 1% crystal violet (Sigma-Aldrich; Merck KgaA, Darmstadt, Germany) for 20 min. Crystal violet-positive invaded cells were counted. Each experiment was performed in triplicate.
2.7. Flow cytometry analysis
The fluorescent antibodies used in the study included fluorescein isothiocyanate (FITC)-conjugated anti-human CD44 and phycoerythrin (PE)-conjugated anti-human CD24 (BD Biosciences, San Diego, CA, USA). IgG of the same isotype and same species was used as an isotype control for each antibody (BD Biosciences, San Diego, CA, USA). Analyses were performed by using a flow cytometer (BD FACSCanto, San Jose, CA, USA).
2.8. Colony formation assay
Single cell suspension was seeded in triplicate at 300 cells per well in six-well plates for 24 h, with the SKOV3 cells treated with diﬀerent concentrations of celastrol (0, 0.25, 0.5, and 1 μΜ) and cultured for 10–14 days. Old media was replaced with fresh media containing ce-lastrol every 3 days. When the cells grew to visible colonies, the media was discarded and the cells were washed with PBS then fixed with 4% paraformaldehyde for 15 min. After the cells were stained with 1% crystal violet for 15 min, the crystal violet was washed out. Plates were photographed and colonies were counted manually. Images are shown as representatives of three independent experiments.
2.9. Spheroid formation assay
SKOV3 cells were plated at a density of 5 × 103 cells per well in ultra-low attachment six well plates (Corning Inc., Corning, NY, USA) and incubated in DMEF/F12 (1:1) supplemented with B-27 (Thermo Fisher Scientific, Grand Island, NY, USA), 10 ng/ml fibroblast growth factor-basic (bFGF, R&D, Minneapolis, MN, USA) and 10 ng/ml epi-dermal growth factor (EGF, Gibco, Carlsbad, CA, USA). The SKOV3 cells were treated with diﬀerent concentrations of celastrol (0, 0.25, 0.5, and 1 μΜ) after plating for 24 h. Every three to four days, half of the medium was replaced. Cells were cultured for 10 days. The generated spheroids were counted and photographed using an inverted micro-scope (CKX71, Olympus Corporation, Tokyo, Japan). The number of spheres with a diameter of > 50 µm was quantified by Image J soft-ware.
2.10. Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted according to the manufacturer's instruc-tions using TRIzol (Thermo Fisher Scientific, Waltham, MA, USA) and then reverse transcription was carried out using a PrimeScript RT kit (Takara Biotechnology Co., Ltd., Dalian, China). SYBR Green qPCR (Takara Biotechnology Co., Ltd., Dalian, China) amplifications were performed in an Applied Biosystems 7500 Fast Real-Time PCR system (Thermo Fisher Scientific, Waltham, MA, USA). The primers used are listed in Table 1. Expression level was normalized by actin. Each ex-periment was repeated three times.