Fig 2: Epithelial-to-mesenchymal states of S18 and S26 clones of CNE-2 cells.(A) Flow cytometry histograms of EMT-associated markers CD90, CD146, CD105, and CD166 in S26 and S18 cells. (B) FACS profiles for CD44 and CD104 of S26 and S18 cells. (C) Relative mRNA expression level vs. GAPDH of EMT transcription factors (EMT-TFs) in S26 and S18 cells (Mean+/- SD of three biological replicates).

Fig 3: E/M state cells exhibit the highest tumorigenicity and plasticity.(A) E, E/M, and xM state cells were sorted from the S26 cells stably expressing LMP1 and LMP2A by FACS. (B) The expression levels of LMP1, LMP2A, CD104, CD44, EMT-TFs (SNAI1, ZEB1), and EMT markers (E-cadherin and Vimentin) in these subpopulations were determined by Western blot. (C) The sorted E, E/M, and xM cells were injected into BALB/c nude mice subcutaneously at limiting dilutions and the numbers of tumor formation were monitored weekly and counted after six weeks. Tumor initiation cell (TIC) frequencies (95% confidence intervals) were calculated using the extreme limiting dilution analysis (ELDA) [47]. (D) Then, mice were sacrificed and tumor weights were measured. (E) Tumors formed by E, E/M, and xM cells were analyzed by H&E staining, and the expression levels and localization of Ki67, LMP1, and LMP2A were analyzed by IHC assay. Ki67 proliferation index was assessed by point counting 500 cells and reported as the percentage of positive cells (Mean +/- SD of three randomly chosen fields under a microscope, 200x). (F) The representative images of IHC are shown. (G) CD44 (red) and CD104 (green) expression profiles in different areas of one tumor sample were analyzed by immunofluorescence assay (630x). (H) FACS profiles of CD44 and CD104 in cells isolated from tumors formed by E, E/M, and xM cells. (I) Three cases of NPC clinical samples were examined by H&E staining and the expression levels of CD44, CD104, LMP1, and LMP2A were detected by IHC assay. Two cases are shown. (J) Immunofluorescence assay to detect the localization and expression level of CD44 (red) and CD104 (green) in different areas of one NPC clinical sample.

Fig 4: LMP2A contributes to EMT by stabilizing the LMP1 level in cells.(A) S26-LMP1-zsgreen stable cells were transduced with pLVX-EF1a-LMP2A-N1-Puro (LMP2A-puro) or CTR-puro lentiviral vectors (CTR-puro) to establish the S26-LMP1zsgreen/2Apuro (S26-LMP1/2A) and its control cell lines (S26-LMP1). The population of LMP1 positive (zsgreen positive) was analyzed by flow cytometry analysis. (B) The expression levels of LMP1 and LMP2A in S26-CTR, S26-LMP1 and S26-LMP1/2A cells were confirmed using Western analysis. (C) The maintenance of LMP1high population (zsgreenhigh) in S26-LMP1 and S26-LMP1/2A cells over time after sorting (up to 12 days) was determined by flow cytometry. (D) The CD44 and CD104 FACS profiles of S26-LMP1 and S26-LMP1/2A cells were analyzed on Day 12. (E) Three EBV positive NPC cells (HK-1+, CNE2+, C666-1) were transduced with LMP2A expression lentiviral vectors or control (pLVX-EF1a-LMP2A-N1-Puro or CTR-puro). Relative mRNA expression levels (relative to GAPDH) of LMP1 and LMP2A in these cells are shown. (Mean+/- SD of three biological replicates). (F) S26-LMP1 cells and S26-LMP1/2A cells were treated with 100 µM cycloheximide (CHX) for 0, 1, 2, 4 hours, and the LMP1 and LMP2A levels were analyzed using Western blot and plotted against time. The data are represented as percentages of the protein detected at the beginning of the incubation (0 hours), all protein signals have normalized to ß-actin.