Figure 1.
Chemical structure of ergosterol.
Figure 2.
The HPLC chromatograms of FPKc (A), standard ergosterol (B).
FPKc and ES standard were identified by HPLC-PDA at 254 nm as described in the experimental section.
Figure 3.
SW-480, SW-620, Caco-2 and HEK-293 cells viability after FPKc (A, B, C, D) and ES (E) treatment was measured by MTT assay. Each value was expressed as a mean ± S. D. of at least three independent determinations. One-way ANOVA was used for comparisons of multiple group means followed by Dunnett’s t-test. *P<0.05 and **P<0.01 versus the control. (error bars = S. D., n = 3).
Figure 4.
Effects of FPKc and ES on the migration of SW-480 cells in vitro. Figure 4A, Detection of cell migration ability after different treatments using wound healing assay.
SW-480 cells in 24-well plates were wounded by scratching with a pipette tip and the cells were incubated with FPKc and ES for 12, 24 hours. The cells were photographed under phase-contrast microscopy (×200 magnification). Figure 4B, Analysis of change in migration on SW-480 cells by transwell assay. Cells in each group move to the lower surface of the filter were stained with crystal violet and photographed under a light microscope at ×200. b) The OD ratio of crystal violet was measured. Error bars represent SD of the means from three independent experiments. *p<0.05 and **p<0.01 versus untreated control.
Figure 5.
Measurement of MMP-2 and MMP-9 expression level in SW-480 cells after FPKc treatment.
SW-480 cells were fixed and processed for immunofluorescence, MMP-9 and MMP-2 were visualized using FITC-label second antibody (green). Scale bars, 100 µm.
Figure 6.
FPKc and ES effects on the cell morphology and nucleus in SW-480 cells.
SW-480 cells treated for 48 h were stained with Hoechst 33342. Morphological changes were observed under fluorescent microscope.
Figure 7.
Effects of FPKc and ES on DNA fragmentation of SW-480 (A) and HEK-293 (B) cells.
Both Cells were treated with FPKc and ES for 12(PI) and analyzed by flow cytometry.
Figure 8.
Cell cycle analysis of FPKc and ES-treated cells.
SW-480 cells were harvested and fixed in 70% alcohol and then stained with PI. Finally the stained cells were analyzed using a flow cytometer.
Figure 9.
FPKc and ES induced apoptosis on SW-480 (A), HEK-293 (B), and SW-620 cells (C).
Cells were double-stained with Annexin V-FITC and PI, and then analyzed by flow cytometry. All experiments were done independently in triplicate per experimental point, and representative results were shown. The results represented the mean±SD of three independent experiments. *p<0.05 and **p<0.01 indicated statistically significant differences versus control group.
Figure 10.
ROS generation triggered by FPKc and ES.
SW-480 (A) and HEK-293 (B) cells were treated with FPKc and ES, and the ROS levels were measured by flow cytometry after staining with DCFH-DA. SW-480 cells were pretreated with NAC (5 mM) for 1 h, then intracellular ROS generation (C), DNA damage (D), cell viability (E) and apoptosis (F) were detected.
Figure 11.
Alterations of cellular GSH levels after treatment with FPKc and ES.
Intracellular GSH concentration of SW-480 cells after FPKc and ES treatments was measured at 405 nm with microplate reader.
Figure 12.
Effects of FPKc (A) and ES (B) on the expression of proteins associated with cell cycle and apoptosis in SW-480 cells.
SW-480 cells were treated with 240 µg/ml FPKc and 24 µg/ml ES for 12, 24, 48 h. Western blot analysis was performed in triplicate per experimental point; Actin was used as reference control.
Figure 13.
Proposed possible signal pathways for FPKc-induced apoptosis and migration inhibition in human colon cancer SW-480 cells.