Tfap2a

Total RNA were isolated from indicated cells as described as above. Single-strand cDNA was synthesized using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., United States). Primer sets for IGFBP2, GREM1 (Gremlin 1, DAN Family BMP Antagonist), BARX1 (BarH-like homeobox 1), and ALPL (alkaline phosphatase, liver/bone/kidney) were purchased from the Integrated DNA Technologies (IDT, Coralville, Iowa., United States). Other primer sets were synthesized by IDT as follows: TFAP2A(transcription factor AP-2 alpha)-For: 5′-GAGCCATGGCACGCACGAGACGGTATCTA-3′, TFAP2A-Rev: 5′-GAGCTCGAGCTCGCAGTCCTCGTACTTGA-3′; LYPD6B (LY6/PLAUR Domain Containing 6B)-For: 5′-GTTTCCTGACCCGTGAAATG-3′, LYPD6B-Rev: 5′-GTCCCGTCCAGATGTTGG-3′; RUNX2-For: 5′-TTACTTACACCCCCCAGTC-3′, RUNX2- Rev: 5′-CACTCTGGCTTTGGG AAGAG-3′;and endogenous control GAPDH-For: 5′-AGGTCGGAGTCA ACGGATTTG-3′, GAPDH-Rev: 5′-GGGGTAACTGTGC-CTATTCG-3′. Quantitative PCR using SYBR Green SuperMix (Qiagen) was performed as we previously described (Lu et al., 2014 (link)). The level of mRNA expression was measured using threshold cycle (CT) according to the ΔΔCT method (Livak and Schmittgen, 2001 (link)).

The promoter fragments of IL1R1 and RAB27B were amplified from genomic DNA by PCR and subcloned into pGL3-Basic (Promega). Primers were listed in Table S4. Dual-luciferase assay was performed according to manufacturer’s instruction (Promega). Luciferase reporters of MYC, EP300, TP53, and TFAP2A were obtained from Qiagen Inc. and Stratagene (La Jolla, CA). Luciferase activity was measured with a luminometer (Lumat LB9507, Berthold, Germany).