microarray.org). DNA microarrays contained 41,125 cDNA clones and represented approximately 24,473 unique genes. The cDNAs were amplified and spotted on glass slides in duplicate by using a robotic arrayer. Total RNA (100μg) was labeled by reverse transcription in the presence of Cy5(red)-labeled or Cy3(green)-labeled GSK-3 activation nucleotides (Amersham Biosciences, Piscataway, NJ). Two labeled RNAs were competitively hybridized to the microarray, and the signals were analyzed
by using a GenePix 4000A scanner (Axon Instruments, Molecular Devices, Palo Alto, CA). Quantitation was performed by using GenePix Pro 5.0 (Axon Instruments). Chromatin immunoprecipitation (ChIP) assay was performed on Hep3B cells, infected
by 75 MOI Ad-PPARγ or Ad-LacZ as control using EZ-Magna ChIP A kit (Millipore, Billerica, MA). Chromatin DNA fragments were precipitated with 10 μg PPARγ antibody (Santa Cruz Biotechnology). The DNA www.selleckchem.com/products/BMS-777607.html was then de-crosslinked and extracted from the DNA-protein complex. The immunoprecipitated DNA was subjected to ChIP-PCR validation. To confirm the presence of PPARγ binding on promoter targets, we performed ChIP-PCR using four known PPARγ-responsive targets, including Acyl-coenzymeA oxidases (ACOX), phosphatase and tensin homolog (PTEN), fibronectin (Fn), and thromboxane A2 receptor (TBXA2R). Immunoprecipitated DNAs were amplified with primers (Supporting Table 1) flanking the consensus sequences of the PPARγ-responsive elements. After confirming that PPARγ binds to the above promoters, we demonstrated the success of ChIP against PPARγ. Expression of growth differentiation factor 15 (GDF15) promoter was detected in ChIP DNA samples with primer sequences listed
in Supporting Table 1. The final PCR products were electrophoresed on 1.5% agarose gels and photographed under ultraviolet light. GDF15 and Ki-67 were detected in paraffin-embedded MCE liver sections using the specific antibodies and an avidin-biotin complex immunoperoxidase method.14 The proliferation index was determined by counting the numbers of cells staining positive for Ki-67 as percentages of the total number of tumor cells. At least 1000 tumor cells were counted each time. Total RNA was extracted from frozen liver tissues and cell pellets with RNA Trizol reagent (Invitrogen, Carlsbad, CA). The messenger RNA (mRNA) expression level of the target gene was determined by reverse transcription PCR (RT-PCR).7 Data were expressed as mean ± standard deviation (SD). Nonparametric data between two groups was computed by chi-squared test or Fisher Exact test. Multiple group comparisons were made by one-way analysis of variance after Bonferroni’s correction or Kruskal-Wallis test where appropriate. The difference for two different groups was determined by Mann-Whitney U test. A P value of less than 0.05 was considered statistically significant.