br Cell viability was also qualitatively evaluated by live d
Cell viability was also qualitatively evaluated by live/dead staining.
A total of 2 × 103 JSH-23 were seeded in 96-well plates with 100 μL complete medium. After exposure to various APS and CM, all cells were treated with calcein-AM, PI and Hoechst 33342 staining solution at each time point and incubated at 37 °C for 30 min. Imaging was per-formed by a confocal laser scanning microscope (FV1000, Olympus, Materials Science & Engineering C 98 (2019) 685–695
Colony formation assay was conducted to evaluate the inhibitory eﬀect of CM on MCF-7 cells. MCF-7 cells in logarithmic phase were seeded at a density of 800 cells/well in 6-well culture plates. After cell adhesion, CM with diﬀerent concentrations was added and the cells were incubated for 2 weeks. Afterwards, cells were stained with Giemsa's stain for the colony count.
MCF-7 cells (1 × 106 cells/mL) were seeded in 6-well culture plates and treated with various concentrations of APS and CM for 48 h. Cells were collected according to the routine digestion by trypsin. Then they were washed with cold PBS and fixation with 70% ethanol at 4 °C for 12 h. Subsequently, cells were treated with 100 μg/mL RNase solution and PI staining at 37 °C for 30 min. The samples were analyzed by a flow cytometer (FACS Canto, BD Biosciences, USA). The relative pro-portions of cells in the G0/G1, S and G2/M phases were analyzed with Modfit software (Verity Software House Inc., Topsham, ME, USA).
2.4.6. Detection of cell apoptosis
MCF-7 cells (3 × 103 cells/well) were seeded in 96-well plates and were treated by various concentrations of APS and CM for 48 h. DAPI (5 μg/mL in PBS) staining was conducted to evaluate the nuclear morphology. Cell apoptosis was evaluated by dual (AO/EB) fluorescent staining . Apoptotic rate of MCF-7 cells was also assessed using an Annexin V–FITC/PI (Sigma, St. Louis, MO) staining assay after exposure to CM for 48 h as previously described .
A total of 20 μL MCF-7 cells (1.5 × 105 cells/mL) were seeded on cover slips and treated with various concentrations of APS and CM for 48 h. Subsequently, cells on the cover slips were fixed with 2.5% glu-taraldehyde at 4 °C for 3 h, post-dehydrated with progressively in-creasing concentrations of ethanol (50%, 70%, 90% and 100%) and dried at room temperature. The specimens were coated with gold and examined with SEM with accelerating voltage of 30.0 kV and exposure time of 6 μs.
Immunofluorescence and flow cytometry assays were conducted to evaluate CM eﬀects on Bcl-2/Bax expression. After 48 h of incubation, the cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS. Subsequently, cells were blocked with 10% normal goat serum for 1 h at room temperature and exposed to human anti-Bcl-2 antibody (rabbit monoclonal antibody, 1:100), anti-Bax an-tibody (rabbit monoclonal antibody, 1:100) overnight at 4 °C. Following the removal of primary antibodies, cells were then incubated with Dylight 549 conjugated goat anti-rabbit antibody (Beyotime, 1:50) for 1 h and the nuclei were counterstained with DAPI. Furthermore, Bcl-2/Bax protein expression was evaluated by flow cytometry as pre-viously described .
2.5. Cell migration and invasion assay
The eﬀects of APS and CM on cell migration and invasion were evaluated in a 24-Transwell cell culture chamber (8 μm, Corning Incorporated, Acton, MA). A total of 300 μL cells (2 × 105 cells/mL) were seeded in the upper chamber in serum-free DMEM culture medium and diﬀerent concentration of APS and CM, while DMEM medium with 10% FBS (600 μL) was added to the bottom well as a chemoattractant. After 24 h incubation, cells on the upper surface of membranes were removed with cotton swabs and the migrated cells on the lower surface were fixed with 4% paraformaldehyde for 10 min and stained with eosin for 20 min. Migrated cells were visualized and
photographed with an inverted phase contrast microscope (Olympus, Japan). For the invasion assays, the experimental procedures were si-milar to that of migration assays except that the Transwell invasion chambers were coated with Matrigel (50 μL per filter) (BD Biosciences, Franklin Lakes, NJ, USA) and the incubation was extended to 48 h. After eosin staining, images of three random fields from three replicate wells were selected and the numbers of cells were calculated.
2.6. Statistical analysis
All data were expressed as the mean ± standard deviation (SD). All experiments were performed at least three times. Significances were analyzed by one-way analysis of variance (ANOVA) and Student's t-test. P < 0.05 was considered as significant diﬀerence. All statistical ana-lyses were performed using Origin 8.0 software (Origin Lab, MA, USA).