Animal experiments were performed according to the guidelines set by the animal safety center, Japan. RT-PCR Total RNA from cells, tumors and normal tissues was isolated using the TRIZOL reagent (Invitrogen) according to the manufacturer’s standard instructions. Reverse transcription was performed with random primers using the High Capacity cDNA reverse transcription kit (ABI). PCR was performed using primers listed in Table 1. These primer sets are applicable to the detection of the messages in mouse ES cells [10]. PCR cycles were usually 35 rounds, and otherwise

selleck described. We avoided quantitative interpretation of the results of RT-PCR analysis. The amplified DNA fragments were analyzed with 1% agarose gel and stained with etidium bromide. Table 1 Primer sequences Primer name Primer sequence (F: forward) Primer sequence (R: reverse) Dppa2 agaagccgtgcaaagaaaaa gttaaaatgcaacgggctgt Fthl17 actttgggactgtgggactg ttgatagcatcctcgcactg Sall4 gcccctcaactgtctctctg gggagctgttttctccactg Rex1 caggttctggaagcgagttc gacaagcatgtgcttcctca Utf1 ttacgagcaccgacactctg cgaaggaacctcgtagatgc Tcl1 caccatgagggacaagacct cttacaccgctctgcaatca Sox2 atgggctctgtggtcaagtc ccctcccaattcccttgtat Dppa3 ctttgttgtcggtgctgaaa tcccgttcaaactcatttcc Gdf3 acctttccaagatggctcct cctgaaccacagacagagca

Ecat8 tgtgtactggcaaccaaaa ctgaggtcccatcagctctc Dnmt3l caagcctcgtgactttcctc ccatggcattgatcctctct Eras atcctaacccccaactgtcc caagcctcgtgactttcctc Fbxol5 ctatgattggctgcgacaga see more Sirolimus nmr gtagtgtcgggaggcaatgt Dppa5 cagtcgctggtgctgaaata tccatttagcccgaatcttg Ecatl gaatgcctggaagatccaaa aaatctcagctcgcctttca Dppa4 agggctttcccagaacaaat

gcaggtatctgctcctctgg Soxl5 cggcgtaagagcaaaaactc tgggatcactctgagggaag Oct3/4 ccaatcagcttgggctagag ctgggaaaggtgtccctgta Nanog cacccacccatgctagtctt accctcaaactcctggtcct c-Myc gcccagtgaggatatcttgga atcgcagatgaagctctggt Grb2 tcaatgggaaagatggcttc gagcatttcttctgccttgg β-catenin gtgcaattcctgagctgaca cttaaagatggccagcaagc Stat3 agactacaggccctcagcaa cctctgtcaggaaaggcttg second CD133 ctcatgcttgagagatcaggc cgttgaggaagatgtgcacc CD24 actctcacttgaaattgggc gcacatgttaattactagtaaagg CD44 gaaaggcatcttatggatgtgc ctgtagtgaaacacaacacc ABCB5 gtggctgaagaagccttgtc tgaagccgtagccctcttta GDF3 aaatgtttgtgttgcggtca tctggcacaggtgtcttcag Quantitative PCR We used the following PCR primers: GDF3-F1, GDF3-R1, β-actin-F1, and β-actin R1 for quantitative PCR. Their sequences for GDF3 gene are listed in Table 1, and those of β-actin are a follows: β-actin-F1: TTT GCA GCT CCT TCG TTG C, and β-actin-R1: TCG TCA TCC ATG GCG AAC T. Quantitative PCR was performed by Step One real-time PCR system (ABI). The statistical comparisons were performed using the Student’s t test between two groups. Tumor transplantation B16 melanoma cells or G1, G5 hepatoma cells were cultured in 10-cm dishes and harvested with 0.02% EDTA solution. Cells were washed two times with D-PBS.