Table 1.
Primers used to quantify mRNA.
Figure 1.
PPAR δ knockdown promoted the growth of xenografts in nude mice.
(A). Growth curve of xenografts. At each time point, the xenografts in PPAR δ-silenced group (n = 12) grew significantly larger than those of control group (n = 12) (*P<0.05), even when treated by bevacizumab (**P<0.05) (mean±SD, t-test). (B). Representative photograph of mice in each group was taken 25 days after inoculation. (C). The xenografts when nude mice sacrificed. (D) The xenografts in PPAR δ-silenced group were significantly heavier than those in control group when nude mice sacrificed (mean±SD; *P = 0.021; **P = 0.02; t-test).
Figure 2.
PPAR δ knockdown induced less differentiation of xenografts.
(A). Representative photographs of xenografts by HE staining. The xenografts in PPAR δ-silenced group (n = 12) were obviously less-differentiated than those in control group (n = 12).×400 magnification. (B). PPAR δ- silenced group had significantly more less-differentiated xenografts than control group (85% vs. 17%, P = 0.025; Chi-square test). (C). Shown by quantitative RT-PCR, the mRNAs encoding ADRP, L-FABP or ALPI were significantly decreased in PPAR δ-silenced group relative to control group (P<0.05).
Figure 3.
The expression of Ki67 in xenografts.
(A). Representative photographs of Ki67 expression in xenografts stained by IHC.×400 magnification. (B). PPAR δ- silenced group (n = 12) had significantly higher score of Ki67 staining than control group (n = 12) (*P = 0.026), even after treated by bevacizumab (**P = 0.031) (mean±SD; t test). (C). The mRNA level of Ki67 was significantly higher in PPAR δ-silenced group than in control group (*P = 0.018), even after treated by bevacizumab (**P = 0.025).
Figure 4.
PPAR δ knockdown had no effect on apoptosis detected by TUNEL assay in xenografts.
(A). Representative pictures of each group. The total cells were identified by DAPI stain, and the positive apoptosis cells were identified by FITC stain.×200 magnification. (B). Quantitative data of apoptosis index (AI). There wasn’t significant difference of AI between groups even after treated by bevacizumab (mean±SD,*P = 0.81, **P = 0.75; t-test).
Figure 5.
PPAR δ knockdown increased VEGF expression in xenografts.
(A). Representative photographs of VEGF expression in xenografts stained by IHC.×400 magnification; (B). PPAR δ- silenced group (n = 12) had significantly higher score of VEGF staining than control group (n = 12) (*P = 0.028; rank sum test). (C). Quantitative RT-PCR result (*P = 0.032).
Figure 6.
KM12C cells secreted less VEGF after PPAR δ activation.
The KM12C cells were treated with serial concentrations of GW501516 or vehicle, and the cell-free supernatants were collected for VEGF quantification by ELISA. Treated by GW501516, the control cells showed a dose-dependent decrease of VEGF secretion (*P = 0.018), while the PPAR δ-silenced cells had no significant change all along (**P = 0.83; ANOVA).