Table 1.
RT-PCR primers sequences and fragment.
Fig 1.
Anti-proliferative activity of FNC on JeKo-1 cells.
Detected by CCK-8 assay after 24, 48 and 72 h treatment. Values are Means±SD (means of three independent experiments). FNC shows a dose-and time-dependent anti-proliferative activity in JeKo-1 cells.
Fig 2.
Effects of FNC on cell cycle distribution and apoptosis.
(A)FNC induces G1/S or G2/M phase cell cycle arrest. JeKo-1 cells were incubated with 0, 0.0625, 0.125, 0.25, 1 μmol/L FNC for 48 h. The cell cycle distribution was determined via flow cytometry. Data are representative of one of three similar experiments. (C)JeKo-1 cells were treated with 0, 0.0625, 0.125, 0.25, 1 μmol/L FNC for 48 h and harvested. Flow cytometry was performed to observe apoptosis rates. Data are representative of one of three similar experiments. (B and D) Quantified histograms display the effect of FNC on JeKo-1 cells cycle distribution and apoptosis. Data are expressed as means ± SD for 3 independent experiments. * P<0.05 versus the control.
Fig 3.
Effect of FNC treatment on Fas, FasL, TNF-α protein expression in Jeko-1 cells.
(A) Western blot of proteins extracted from Jeko-1 cells following 48h treatment with FNC (0μM, 0.0625μM, 0.125μM, 0.25μM and 1.000μM). A representative result of 3 independent experiments is shown. (B) The Fas/GAPDH, FasL/GAPDH and TNF-α/GAPDH ratio is displayed as Mean±SD. * P<0.05 versus the control.
Fig 4.
FNC significantly inhibited the growth of human JeKo-1 xenografts in vivo.
(A)Graphs represent the average weight of tumors from every group. (B)shows the average proliferation index(proliferation index% = (tumor weight of drug group/tumor weight of negative control group)×100%]. (C) Graphs represent the average body weight, the body weights were measured after treatment. Tumor weights and body weights are presented as means±SD. * P<0.05 versus the control.
Fig 5.
Histopathological examination of liver and kidney in FNC treated mice.
When the mice were euthanized, liver and kidney were collected and fixed in 4% buffered paraformaldehyde and paraffin embedded for H&E staining. Pictures were original captured at 100× magnification.
Table 2.
Table analysis of GO enrichment.
Table 3.
The main GO terms of cellular process.
Table 4.
Positive regulation of cell proliferation.
Table 5.
Negative regulation of cell proliferation.
Table 6.
Cell cycle arrest related genes covered in GO.
Table 7.
Genes related to apoptosis.
Fig 6.
Gene expression profiling analysis of differential expression genes in xenograft tissue with and without FNC treatment.
Red color represents the up-regulated genes, and green color represents the down-regulated genes. (A)Hierarchical clustering(p≤0.05, fc≥2 or fc≤0.5, mean = 7). (B)Volcano plot. The x axis represents the differential expression profiles with the fold-induction ratios in a log2 scale, and the y axis represents the P value of T-test in a log10 scale. Differentially expressed genes are established at Fold change ≥2 and P < 0.05.
Fig 7.
Pie chart of pathway enrichment.
We screen out the top 10 significant enrichment pathway terms based on Biocarta. The percentage of each pathway is shown.
Fig 8.
Verification of microarray data by RT-PCR.
(A) Electrophoresis image of agarose gel. GAPDH was used as an internal control. The pictures shown are representatives of 3 independent experiments. (B)Gene expression levels of 5 selected genes. The right panel of each gene shows the gene expression levels as detected by RT-PCR, and the left panel of each gene shows the gene array data. It shows that the result of RT-PCR was consistent with the microarray results.