BCBL-1 cells were treated with a final concentration of 1% formaldehyde and crosslinked for 10 min at RT. Crosslinking was stopped by addition of glycine at a final concentration of 0.125 M for 5 min. The cross-linked cells were washed in 16 PBS and counted so that approximately 10 7 cells were used in each immunoprecipitation (IP) reactions. Nuclei from the cells were purified and lysed to collect chromatin. Chromatin was sheared to approximately 500 bp using a Bioruptor sonicator (Diagenode, Sparta, NJ). Lysates containing the chromatin were pre-cleared using 35 ml of Protein A sepharose beads (Amersham Biosciences) in pre-IP dilution buffer for 30 min at 4uC. The samples were centrifuged to remove beads and the lysate was recovered. After setting aside input controls, primary antibodies were added to the samples and incubated overnight at 4uC. The DNA/protein complexes were IP using protein A beads for 4 h at 4uC and then washed using various ChIP wash buffers. Following elution, proteinase K was added to the complexes and incubated at 65uC overnight in order to reverse the crosslinks. The DNA samples were purified using phenol/chloroform extraction, resuspended in 16 TE buffer, and finally analyzed by polymerase chain reaction (PCR). PCR was performed using platinum pfx polymerase (Invitrogen, Carlsbad, CA) as per standard protocols. PCR amplification of the precipitated DNA was performed using primers that targeted ORF50P3, ORF50P8, and ORF50PNP regions ( Table 1) Figure S1 Resveratrol inhibits Egr-1 expression in the absence of KSHV infection. HEK293 cells were mockinfected by incubating with growth medium for 2 h at 37uC. These cells were washed and cultured in growth medium in the presence or absence of 100 mM of resveratrol for 48 h. The cells were lysed using gold lysis buffer (GLB) and the lysates were resolved on a 10% SDS-PAGE, transferred to a PVDF membrane, and Western blotting was performed using specific antibodies. (TIF) Common Variants in CDKN2B-AS1 Associated with Optic-Nerve Vulnerability of Glaucoma Identified by Genome-Wide Association Studies in Japanese BACKGROUND: To date, only a small portion of the genetic variation for primary open-angle glaucoma (POAG), the major type of glaucoma, has been elucidated. METHODS AND PRINCIPAL FINDINGS: We examined our two data sets of the genome-wide association studies (GWAS) derived from a total of 2,219 Japanese subjects. First, we performed a GWAS by analyzing 653,519 autosomal common single-nucleotide polymorphisms (SNPs) in 833 POAG patients and 686 controls. As a result, five variants that passed the Bonferroni correction were identified in CDKN2B-AS1 on chromosome 9p21.3, which was already reported to be a significant locus in the Caucasian population. Moreover, we combined the data set with our previous GWAS data set derived from 411 POAG patients and 289 controls by the Mantel-Haenszel test, and all of the combined variants showed stronger association with POAG (P<5.8×10(−10)). We then subdivided the case groups into two subtypes based on the value of intraocular pressure (IOP)—POAG with high IOP (high pressure glaucoma, HPG) and that with normal IOP (normal pressure glaucoma, NPG)—and performed the GWAS using the two data sets, as the prevalence of NPG in Japanese is much higher than in Caucasians. The results suggested that the variants from the same CDKN2B-AS1 locus were likely to be significant for NPG patients. CONCLUSIONS AND SIGNIFICANCE: In this study, we successfully identified POAG-associated variants in the CDKN2B-AS1 locus using a Japanese population, i.e., variants originally reported as being associated with the Caucasian population. Although we cannot rule out that the significance could be due to the differences in sample size between HPG and NPG, the variants could be associated specifically with the vulnerability of the optic nerve to IOP, which is useful for investigating the etiology of glaucoma. Glaucoma is a neurodegenerative ocular disease and one of the leading causes of irreversible blindness worldwide [1] . It is characterized by the progressive loss of retinal ganglion cells and optic nerve axons, resulting in visual field defects [2] . One of the well-known major risk factors for glaucoma is elevated intraocular pressure (IOP) [2] . Thus, the measurement of IOP is routinely involved in the diagnosis of glaucoma. In fact, the IOP level has been applied to subdivide the most common form of glaucoma, primary open-angle glaucoma (POAG), into two subtypes [3] : POAG with high ($22 mmHg) IOP (POAG/HPG, high pressure glaucoma; hereafter referred to as ''HPG'') and with normal (,22 mmHg) IOP (POAG/NPG, normal pressure glaucoma; hereafter referred to as ''NPG''). Interestingly, ,92% of the Japanese POAG patients are categorized into the NPG subtype [4] , whereas ,41% of Caucasian POAG patients are categorized as NPG [5] , thus showing a unique epidemiological distribution of Japanese patients compared with other ethnic groups.