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PTB-Associated Splicing Factor (PSF) Is a PPARγ-Binding Protein and Growth Regulator of Colon Cancer Cells

Figure 5

Real-time PCR measurement of PPARγ mRNA expression in 4 colon cancer cell lines.

(A) The relative PPARγ levels (HT-29, DLD-1, Caco-2, and LOVO). normalized to 18S rRNA are expressed as mean ± SEM (n = 3), **P<0.01. (B) Representative western blot of PPARγ1 expression. Cell lines were separated into nuclear and cytoplasmic fractions, and 50 µg of protein from the cytoplasmic fraction was analyzed by SDS-PAGE, western blotted, and visualized with enhanced chemiluminescence as described in the Materials and Methods section. (C) and (D) Effect of rosiglitazone on reporter activation in colon cancer cells. Cells were transiently transfected with a pGL3-PPRE-acyl-CoA oxidase luciferase reporter vector or pcDNA3.1-PPARγ vector. The cells were treated with 10 µM rosiglitazone for 20 h. Luciferase activity was normalized to Renilla luciferase activity. Data are expressed as mean ± SEM (n = 4), **P<0.01. (E) Cells were transfected with expression plasmids encoding FLAG-PPARγ and siRNA PSF for 72 h. Next, 10 µL of Cell Counting Kit-8 was added to the medium and incubated for 2 h in an incubator with 5% CO2. The amount of orange formazan dye generated was calculated by measuring the absorbance at 450 nm in a microplate reader. Data are expressed as mean ± SEM (n = 4), **P<0.01.

Figure 5

doi: https://doi.org/10.1371/journal.pone.0058749.g005