Histochemistry, and immunohistochemistry staining of xenografts.

<p>A-C: H&E staining of derived xenografts 120 h after inoculation with 1×10⁶ 5-8F cells or primary tumors. Representative images are shown. D-F: IHC staining of human CK34βE12. Magnification, ×400.</p

Quantification of disseminating NPC cells in CAM.

<p>Number of circulating 5-8F and 6-10B cells in the lung and heart of developing chicken evaluated by β-globin–based qPCR and expressed as mean ± SD (n = 5 for each cell line). * <i>P</i><0.05.</p

2D and 3D analysis of invasion and metastasis of NPC cells 48 h after inoculation.

<p>A: Invasion of HONE1-GFP cells through the basement membrane and toward the lower site of CAM, especially in 3D imaging. Invasive depth is shown in the Z-axis. B: Metastasis of HONE1-GFP cells via the blood system. Representative bright-field (DIC), green fluorescent (GFP) and overlay (Merge) images. Magnification, ×40.</p

Tumor xenografts in chorioallantoic membranes (CAMs) inoculated with NPC cells or tumor biopsies.

<p>CAMs inoculated with 0.5×10⁶ or 1×10⁶ 5-8F cells and NPC primary tumors at 24 and 120 h after inoculation. Representative data are shown.</p

Clinical features of NPC patients participated in this study.

<p>^a: NPC patients involved in this study.</p><p>^b: M, male; F, female.</p><p>^c: WHO histopathological classification of NPC (2005), A: keratinizing squamous cell carcinoma; B1: differentiated non-keratinizing carcinoma; B2: undifferentiated non-keratinizing carcinoma; C: Basaloid squamous cell carcinoma.</p><p>^d: The TNM clinical classification for NPC according to AJCC staging, 7th Edition. T-Primary tumor; N-Regional lymph nodes; M-Distant metastasis.</p><p>Clinical features of NPC patients participated in this study.</p

Additional file 2: Figure S1. of RERG suppresses cell proliferation, migration and angiogenesis through ERK/NF-κB signaling pathway in nasopharyngeal carcinoma

Analysis of RAS type GTPase family genes in nasopharyngeal carcinoma primary tumors and nasopharyngeal epithelial tissues. (A, B) Heat map of RAS type GTPase family in NPC (n = 7) and NNE (n = 5) tissues. (A) RAS type GTPase family genes expression alerted using cDNA microarray. (B) Hypermethylated genes of RAS type GTPase family by methylated-DNA capture sequencing. (C) Genes of RAS type GTPase family which were significantly downregulated in cDNA microarray and hypermethylated in methylated-DNA capture sequencing. Methods for methyl-capture sequencing and gene expression array were described in Additional file 4: Supplementary methods. (TIF 2684 kb

Multiplex Methylation-specific PCR analysis in NPC cell lines and xenograft.

<p>All of the cell lines and xenograft showed aberrant methylation on at least one of the two TSGs included in the MMSP assay. The presence of EBV can be only detected in the C666-1 cell line and the EBV-positive xenograft C15. Unmethylated-<i>LMP1</i> can be only detected in LMP1 expressing xenograft, C15. The other cell lines demonstrated negative bands for <i>EBNA1</i> and <i>LMP1</i> markers due to loss of EBV during cell passage.</p

Additional file 3: Figure S2. of RERG suppresses cell proliferation, migration and angiogenesis through ERK/NF-κB signaling pathway in nasopharyngeal carcinoma

Analysis of VEGF and SASP factors in vitro and in vivo. (A) VEGF in RERG-transfected and empty-vector-transfected NPC cells (HK1, C666-1) were determined by western blotting (n = 3). (B) mRNA expression of RERG, IL8, IL1β and TNFα in xenografts of nude mice was determined by qRT-PCR (n = 8). GAPDH was used as an internal control. (C) IHC analyses of the expression of VEGF in tumors from nude mice. Original magnification is × 200. Bar represents 50 μm. Data are shown as means ± SD. **: P < 0.01, ***: P < 0.001 by Student’s t-test or Mann-Whitney U test. (JPG 1577 kb

Nasopharyngeal EBV DNA load and serum VCA/IgA titers in seropositive high-risk population.

<p>EBV, Epstein-Barr virus; VCA, viral capsid antigen; SD, standard deviation; (+), positive; (-), negative.</p><p>Nasopharyngeal EBV DNA load and serum VCA/IgA titers in seropositive high-risk population.</p

Additional file 1: Table S1. of RERG suppresses cell proliferation, migration and angiogenesis through ERK/NF-ÎşB signaling pathway in nasopharyngeal carcinoma

List of primary antibodies used in the study. (DOCX 16 kb