AP-2a overexpression construct was made by PCR amplification of the complete AP-2a cDNA sequence, using primers with EcoRV and SalI ends. Blunt-end ligation was performed using pBABE following BamHI digestion and Klenow polymerase reaction. DNA isolation DNA was isolated using the DNA QiaAmp kit (Qiagen) according to the manufacturer’s protocol. Trichostatin A (TSA) treatment The SCC22B cell line was treated with either 300 nM or 5 mM TSA for 24 hours. The cells were then collected for western blot analysis and DNA isolation. The 5 mM treated cells were used for the bisulfite sequencing analysis. 5-aza-29deoxycytidine treatment Cells were treated with 1 mM of the drug (Sigma-Aldrich) for 96 hours while maintaining ,50% confluency and changing the drug daily. Western blot analysis Whole cell lysates were suspended in lamaelli buffer and boiled for 15 minutes, followed by centrifugation. Lysates were loaded onto a 7.5% acrylamide gel and run for 2 hours at 110 volts followed by semidry transfer of the proteins to nitrocellulose membranes. The membranes were then blocked in 5% milk for 1 hour. 1:1,000 dilution of the primary antibodies were incubated with the blot overnight shaking at 4 degrees Celsius. A one hour incubation with the secondary antibodies (1:5,000 dilution) followed. The blots were then washed in 1x TBS-Tween for 1 hour and films exposed using ECL solutions. Western blots are representative of three independent experiments. MSI analysis Commonly used MSI primers were utilized for the analysis (Bat25, Bat26, Bat40, D2S123, D17S250, D18S55, D18S56, D18S67, and D18S487)[30,31,32,33]. DNA was isolated from HNSCC SCC22B cells with and without AP-2a downregulation using the Qiagen DNA mini kit according to the standard protocol (Qiagen). 5 ng of this DNA (1 ml) was used for PCR along with 10 ml HotStart mix (Qiagen), 6 ml water, and 2 ml primer (20 pmol). The PCR products were subsequently subjected to electrophoresis on an 8% polyacrylamide gel. Combined Bisulfite Restriction Analysis (COBRA) COBRA was performed as previously described [34]. Primer sequences will be provided upon request. Bisulfite sequencing analysis Bisulfite sequencing was performed as previously described [35]. Primer sequences will be provided upon request. Chromatin immunoprecipitation analysis ChIP analysis was done as previously described [34], according to the Upstate Cell Signaling Solutions protocol. Sequences of the References AP-2a Targets Methylation primers used for the quantitative ChIP PCRs can be provided upon request. Hha I digest Global methylation status was analyzed in SCC22B cells +/2 AP-2a shRNA. 1ug of DNA was incubated with 1 unit HhaI (New England Biolabs) at 37 degrees Celsius for 3 hours. The digests were then loaded onto a 1% agarose gel for comparison. Supporting Information Table S1 Found at: doi:10.1371/journal.pone.0006931.s001 (0.08 RTF) MB Figure S1 Diagram of 5 genes displaying the amplified region in respect to the corresponding gene and associated CpG island. Bisulfite sequencing analysis was performed on five genes which yielded differences in methylation following AP-2a downregula- tion in HNSCC. The diagram shows the location of the bisulfite PCR product, the CpG island, and the gene layout. The numbers provided are in respect to the transcription start site. Found at: doi:10.1371/journal.pone.0006931.s002 (2.55 MB DOC) Figure S2 Some methylated genes in HNSCC are unaffected by AP-2a downregulation. Bisulfite sequencing analysis was per- formed on three genes which yielded no substantial differences in methylation following AP-2a downregulation. Solid circles repre- sent methylated CpGs; whereas, open circles represent unmethy- lated CpG sites. Found at: doi:10.1371/journal.pone.0006931.s003 (2.07 MB TIF) Figure S3 AP-2a downregulation in colon cancer cell lines causes a subtle decrease in RASSF1A and PTEN methylation. a) Western blot analysis showing AP-2a downregulation in HT29 and HCT116 cell lines. b) Bisulfite sequencing analysis on MLH1 shows little methylation in WT HCT116 and HT29 cell lines. c) Bilsulfite sequencing analysis on RASSF1A and PTEN in HCT116 and HT29 colon cancer cell lines with and without AP-2a downregulation. Solid circles represent methylated CpGs; whereas, open circles represent unmethylated CpG sites. Found at: doi:10.1371/journal.pone.0006931.s004 (1.67 MB TIF) Acknowledgments We thank Dr. Thomas Carey for the provision of the HNSCC cell lines used in this study, and members of the Eng lab, and Drs. Aaron Gruver, Angela Ting, and Micheala Aldred for thoughtful discussions. CE is the Sondra J. and Stephen R. Hardis Chair of Cancer Genomic Medicine at the Cleveland Clinic, a Doris Duke Distinguished Clinical Scientist, and an American Cancer Society Clinical Research Professor. Author Contributions Conceived and designed the experiments: KB CE. Performed the experiments: KB. Analyzed the data: KB. Contributed reagents/ materials/analysis tools: TR. Wrote the paper: KB. Helped offer critique of experiments: TR. Suggested some experiments and also offered sufficient feedback on the written portion: CE. 1. Damberg M (2005) Transcription factor AP-2 and monoaminergic functions in the central nervous system. J Neural Transm 112: 1281– 1296. 2. Jiang MS, Lane MD (2000) Sequential repression and activation of the CCAAT enhancer-binding protein-alpha (C/EBPalpha) gene during adipogenesis. Proc Natl Acad Sci U S A 97: 12519–12523. 3. Han CY, Lim SC, Choi HS, Kang KW (2008) Induction of ErbB2 by ultraviolet A irradiation: potential role in malignant transformation of keratinocytes. Cancer Sci 99: 502–509. 4. Imagawa M, Chiu R, Karin M (1987) Transcription factor AP-2 mediates induction by two different signal-transduction pathways: protein kinase C and cAMP. Cell 51: 251–260. PLoS ONE | www.plosone.org 7 September 2009 | Volume 4 | Issue 9 | e6931