Fig. 3.

Down-regulation of PPARG reduces proliferation and invasion in vitro and lymph node metastasis in an in vivo prostate orthograft model. (A) Immunoblotting for PPARG, FASN, and ACC in PC3 cells demonstrating reduction of PPARG protein expression following siRNA, along with its effects on FASN and ACC expression. (B) Following siRNA-mediated knockdown of PPARG expression (controlled with NTsiRNA), PC3 cells were functionally assessed using (B) WST-1 proliferation assay, demonstrating reduced growth (n = 3, error bars represent SEM, *P < 0.01; Mann–Whitney), and (C) wound scratch assay, demonstrating reduced migration (n = 3, error bars represent SD, P < 0.001; ANOVA). (D) ELISA-based PPARG transcription reporter assay demonstrating that GW9662 treatment (20 and 50 μM) reduced PPARG transcriptional activity in PC3 and PC3M cells compared with DMSO controls (n = 3, error bars represent SEM, *P < 0.01; Mann–Whitney). Dotted red line signifies the background signal level. (E and F) Representative IHC staining and (G) boxplot of quantification of Ki-67 staining between vehicle control and GW9662-treated PC3 orthotopic prostate tumors (n = 6 vs. 6, 20× magnification, three fields per mouse, *P < 0.0001; Mann–Whitney). (H and I) Representative IHC staining and (J) boxplot quantification of FASN staining between vehicle control and GW9662-treated PC3 orthotopic prostate tumors (n = 6 vs. 6, 20× magnification, three fields per mouse, *P = 0.0004; Mann–Whitney). (K and L) Representative IHC staining and (M) boxplot quantification Pan-CK staining in the lymph nodes between vehicle control and GW9662-treated PC3 orthograft-bearing mice (n = 6 vs. 6, 20× magnification, three fields per mouse, *P < 0.0001; Mann–Whitney). (Red bar, 200 µm.)