Fig 2: 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.

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: Effects of LMP1 and LMP2A expression on the generation of distinct subpopulations in the epithelial-mesenchymal spectrum.(A) The changes of CD44 and CD104 profiles of S26 cells in response to LMP1 and LMP2A expression revealed by flow cytometry analysis. (B) The expression of EMT markers (E-cadherin, Vimentin) and EMT-TFs (SNAI1, Twist, ZEB1) in S26-CTR, S26-LMP1, and S26-LMP2A cells were analyzed by Western blot. ß-actin was included as a loading control. (C) Immunofluorescence assay of S26 cells (S26-CTR) and S26 cells expressing LMP1 (S26-LMP1) or LMP2A (S26-LMP2A) cells for the expression and subcellular localization of E-cadherin, Vimentin, and ZEB1 (630x). (D) S26 cells were transfected with a pLVX-EF1a-IRES-Zsgreen1-LMP1 plasmid that simultaneously expresses LMP1 and Zsgreen from a single mRNA transcript. LMP1high and LMP1low subpopulations were separated and isolated by FACS based on the fluorescence intensity of Zsgreen. (E) The LMP1 and ZEB1 expression levels in these two subpopulations were analyzed by Western blotting. (F) The epithelial-to-mesenchymal states of LMP1high and LMP1low subpopulations were determined on their CD44 and CD104 expression profiles. (G) S26-LMP1 and S26-LMP2A cells were transfected siRNAs to knock down the expression of LMP1 and LMP2A respectively. The knockdown efficiency of siRNAs was analyzed by Western blot. (H) The effects of LMP1 and LMP2A knockdown on CD44 and CD104 profile were analyzed by flow cytometry analysis.