Table 1.
Target primer sequences used in this study.
Figure 1.
Gene expression of Bcl-2 and IAP family in 30 dogs with histiocytic sarcoma.
mRNA expression levels of Bcl-2 and IAP family members in samples collected from 30 dogs with histiocytic sarcoma were analyzed using real-time reverse transcription-polymerase chain reaction (qRT-PCR). Expression levels of each gene were normalized to those of the same gene in canine fibroblasts. Each bar represents the mean ± SE from 3 separate experiments. *p<0.05 and **p<0.01 for survivin vs. the other anti-apoptotic genes (Dunnett’s test).
Figure 2.
Expression of survivin mRNA in cell lines after transfection with siRNA.
Expression levels of survivin mRNA in canine histiocytic sarcoma (CHS) cell lines were analyzed using qRT-PCR at 0, 12, 24, 48, and 72 h after transfection with siRNA, and 0 h point showed basal level of survivin mRNA in each CHS cell line before transfection. Expression levels of each gene were normalized to those of the same target gene in canine fibroblasts. Each bar represents the mean ± SE from 3 separate experiments. Data were statistically analyzed by one-way ANOVA followed by post-hoc test.
Figure 3.
Expression of survivin protein in cell lines after transfection with siRNA.
Survivin protein expression in CHS cell lines was evaluated by western blotting at 48 h after transfection with scrambled and survivin siRNA.
Table 2.
Information on cell lines used in this study.
Figure 4.
Influences of apoptosis after transfection with siRNA.
Apoptotic cells in CHS cells and canine fibroblasts were evaluated using annexin V fluorescent staining at 48 h after transfection with scrambled and survivin siRNA (400 × magnification).
Figure 5.
Imaging of the apoptotic cells after transfection with siRNA.
Imaging of light microscope in CHS cell lines were performed by Wright-Giemsa staining at 48 h after transfection with survivin siRNA (1000 × magnification). Black arrows (?) showed nuclear fragmentation of CHS cells, which was observed at late-stage of the apoptotic process.
Figure 6.
Influences of cell viability after transfection with siRNA.
Cell viability was evaluated by methylthiazole tetrazolium (MTT) assay in CHS cell lines and canine fibroblasts at 24 or 48 h after transfection with scrambled and survivin siRNA. Each bar represents the mean ± SE from 3 separate experiments. *p<0.05 and not significant; N.S. vs. control cells (Post-hoc test).
Table 3.
50% inhibitory concentrations of lomustine and doxorubicin in CHS cell lines.
Figure 7.
Expression of chemoresistance genes in cell lines after transfection with siRNA.
The mRNA expression levels of chemoresistance genes, including ATP-binding cassette transporter B1 (ABCB1), ATP-binding cassette transporter C2 (ABCB2) and O (6)-methylguanine-DNA methyltransferase (MGMT), in CHS cells lines were evaluated at 48 h after transfection with scrambled or survivin siRNA. Expression levels of each gene were normalized to those of the same gene in untreated CHS cells (control) and are represented as the relative expression (% of control). Each bar represents the mean ± SE from 3 separate experiments. *p<0.05 and N.S. vs. control (Dunnett’s test).
Figure 8.
Expression of ABCB1 protein in cell lines after transfection with siRNA.
ABCB1 protein expression in CHS cell lines was evaluated by western blotting at 48 h after transfection with scrambled or survivin siRNA.
Figure 9.
Influences of ABCB1 function in cell lines after transfection with siRNA.
P-glycoprotein (ABCB1) pump activity was assessed by staining with Hoechst-33342 using fluorescence imaging in CHS cell lines and canine fibroblasts at 48 h after transfection with survivin siRNA (400× magnification).
Figure 10.
Influences of phagocytic activity after transfection with siRNA.
Phagocytic rate of latex beads in CHS cell lines was evaluated at 48 h after transfected with scrambled and survivin siRNA as compared to that of untreated cells (pre). Total number of phagocytosed latex beads in 2,000 randomly selected, viable CHS cells was measured using an image analyzer. The relative phagocytic rate (% of pre) is shown. Each bar represents the mean ± SE from 3 separate experiments. *p<0.05 and N.S. pre (Dunnett’s test).