1. Volume 146, Issue 7, Pages 1714-1726.e5 (June 2014)
Increased Levels of the Long Intergenic Non–Protein Coding RNA POU3F3 Promote DNA Methylation in Esophageal Squamous Cell Carcinoma Cells 
Wei Li, Jian Zheng, Jieqiong Deng, Yonghe You, Hongchun Wu, Na Li, Jiachun Lu, Yifeng Zhou 
Gastroenterology 
Volume 146, Issue 7, Pages e5 (June 2014)
DOI: /j.gastro
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2. Figure 1
Linc-POU3F3 physically associates with EZH2. (A) Cellular characterization of linc-POU3F3, the levels of nuclear control transcript (U6), cytoplasmic control transcript (glyceraldehyde-3-phosphate dehydrogenase [GAPDH] mRNA), and linc-POU3F3 were assessed by qRT-PCR in nuclear and cytoplasmic fractions. Data are presented as a percentage of U6, GAPDH, and linc-POU3F3 levels and total levels for each were taken as 100%. Error bars are representative of SD (n = 4). (B) RIP experiments were performed using the EZH2 antibody to immunoprecipitate and a primer to detect linc-POU3F3. (C) RIP enrichment was determined as RNA associated with EZH2 immunoprecipitation relative to the input control. Ab, antibody.
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3. Figure 2
Linc-POU3F3 regulates the POU3F3 gene methylation level in both ESCC cells and tissues. (A) The linc-POU3F3 and POU3F3 gene expression levels in ESCC cells after linc-POU3F3, linc-POU3F3-shRNA, or control lentiviruses infection. Data shown are the mean ± SE of 3 independent experiments, normalized to glyceraldehyde-3-phosphate dehydrogenase. (B) Gene location, amplicon size, and place of CpG sites in the amplicon. Methylation profile of CpG sites for the POU3F3 gene. The color of the circles is related to the percentage of methylation in each CpG site. Boxes indicate the different methylation patterns between linc-POU3F3 up-regulated ESCC cells, control cells, linc-POU3F3 down-regulated ESCC cells, and 3-deazaneplanocin A (DZNep)-treated ESCC cells. (C) Western blot of EZH2 level in Eca-109 and TE-1 cells (human ESCC cells) after DZNep treatment. (D) Three-dimensional contours of standardized log2-transformed linc-POU3F3 expression, POU3F3 methylation level, and POU3F3 gene expression in a set of randomly selected 32 ESCC tumor and paired adjacent non-neoplastic tissues (x-axis relative to linc-POU3F3 expression level; y-axis relative to POU3F3 gene expression level; and heat spectrum relative to POU3F3 methylation level). (E) The POU3F3 gene expression levels in ESCC cells after DZNep treatment. Data shown are the mean ± SE of 3 independent experiments, normalized to glyceraldehyde-3-phosphate dehydrogenase. (F) ChIPs show that association of DNA methyltransferases with POU3F3 depends on the presence of EZH2. Cross-linked chromatin from Eca-109 and TE-1 cells with or without DZNep treatment was immunoprecipitated with the indicated antibodies.
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4. Figure 3
Linc-POU3F3 is involved in the regulation of Notch signaling in vivo. (A) Known and predicted protein–protein interactions in which POU3F3 is involved (obtained from STRING, a database of known and predicted protein interactions). (B) Association between log2-transformed linRNA-POU3F3 and DLL1 expression in a set of randomly selected 32 ESCC tumor and paired adjacent non-neoplastic tissues. (C) Alignment of the DeltaM enhancer sequences from the human DLL1, mouse Delta1, chick Delta1, and zebrafish DeltaD genes. Conserved residues are shown in black, and nonconserved residues are shown in gray. Blue box delineates the conserved octamer binding site of POU3F3. The dashed line outlines the deleted sequence from DeltaM to generate DeltaN that was used in the luciferase reporter assay. (D) Luciferase reporter assay in Eca-109 and TE-1 cells after being transfected with linc-POU3F3, linc-POU3F3-shRNA, or control lentiviruses and the reporter constructs expressing the luciferase gene under the DeltaM enhancer or the defected DeltaN segment. Data are presented as the mean ± SD of quadruplicate assays. (E) Immunofluorescence analysis of DLL1 in Eca-109 and TE-1 cells after transfection with linc-POU3F3, linc-POU3F3-shRNA, or control lentiviruses. (F) Western blot of Notch signaling–associated proteins, POU3F3, DLL1, NICD, HES1, and P21 in Eca-109 and TE-1 cells after transfection with linc-POU3F3, linc-POU3F3-shRNA, or control lentiviruses. DAPI, 4′,6-diamidino-2-phenylindole.
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5. Figure 4
Effects of ectopic linc-POU3F3 expression on ESCC cell cycle and proliferation. (A) Cell-cycle analysis of Eca-109 and TE-1 cells after transfection with linc-POU3F3, linc-POU3F3-shRNA, or control lentiviruses. (B) Summarized flow cytometry data. Results are represented as the average ± SE based on 3 independent experiments (*P < .05 when compared with control). (C) Annexin V–FITC/PI apoptosis assay of Eca-109 and TE-1 cells after transfection with linc-POU3F3, linc-POU3F3-shRNA, or control lentiviruses (*P < .05 when compared with control). (D) Eca-109 and TE-1 cells were seeded in 96-well plates after transfection with linc-POU3F3, linc-POU3F3-shRNA, or control lentiviruses, and cell proliferation was assessed daily for 4 days using the Cell Counting Kit-8 assay (*P < .05 when compared with control).
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6. Figure 5
Effects of ectopic linc-POU3F3 expression on ESCC cell colony formation and tumor growth in vivo. (A) Representative colony formation assay in Eca-109 and TE-1 cells after transfection with linc-POU3F3, linc-POU3F3-shRNA, or control lentiviruses. (B) Quantitative analysis of colony formation. The numbers of colonies in vector-transfected controls were set to 100%. Values are expressed as mean ± SD from 3 experiments (*P < .05 when compared with control). (C-E) Subcutaneously implanted linc-POU3F3–down-regulated (Eca-109-shRNA and TE-1-shRNA cells), linc-POU3F3–up-regulated (Eca-109-linc-POU3F3 and TE-1-linRNA-POU3F3 cells), and respective control cell xenografted tumors were established. Bars show SD. The mean tumor volume from 8 nude mice of each group are shown (*P < .05 when compared with control, photographs of mice used in the Figure were taken at day 21).
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7. Supplementary Figure 1
Linc-POU3F3 recruits EZH2 to the genomic location to regulate POU3F3 gene CpG methylation, in the context of the PRC2/3 complexes, through direct physical contact with DNA methyltransferases, and thus affects Notch signaling.
Gastroenterology  , e5DOI: ( /j.gastro )
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8. Supplementary Figure 2
qRT-PCR analysis in both LMD-measured and non–LMD-measured samples (n = 10).
Gastroenterology  , e5DOI: ( /j.gastro )
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