Fig 2: Lnc-EGFR prevents the ubiquitination of EGFR by binding to Tyr1045.(a) T cells isolated from peripheral blood of HCC patients were transduced with indicated lentiviral particles and then treated with with EGF (20 ng ml-1) for indicated timepoints followed by western blotting for p-EGFR(Y1045), p-EGFR(Y1068), p-EGFR(Y1073), p-ERK1/2(T202/Y204), EGFR, ERK1/2 and ß-actin. (b) Normal, healthy human T cells transduced with mock or indicated lentiviral particles were determined with real-time PCR (upper) and were further treated with EGF (100 ng ml-1) for 90 min or left untreated. Whole-cell lysates were prepared and EGFR was immunoprecipitated followed by western blotting for ubiquitin. Equal loading of EGFR was determined by western blotting via anti-EGFR antibodies (lower). (c) Whole-cells lysates were prepared and c-CBL was immunoprecipitated via anti-c-CBL antibody. The presence of EGFR in the immunecomplex was determined by western blotting via anti-EGFR antibody. (d) Transduced T cells were treated with anti-CD3/anti-CD28 beads (bead-to-cell ratio of 1:1), EGF (20 ng ml-1) or left untreated in the presence or absence of PD98059 (40 µM) and/or CsA (1 µM). Whole-cell lysates were prepared and subjected to Western blotting for p-ERK1/2(T202/Y204), p-MEK1/2(S217/221), p-NF-AT1(S54), ERK1/2, IL-2, MEK1/2 and ß-actin. Each experiment was performed triplicated.

Fig 3: Syncytin-1 promotes doxorubicin resistance via MEK/ERK pathway in HCC.a CCK-8 assay. The effect of doxorubicin on cell viability was determined in HCCLM3 cells. b The regulation of doxorubicin on the expression of MEK/ERK pathway protein (MEK1/2, ERK1/2, p-MEK1/2, p-ERK1/2, CCND1, and CDK4) were detected by western blotting. c CCK-8 assay was used to investigate the role of Syncytin-1 on doxorubicin-induced cell apoptosis in Huh7 cells. d The function of Syncytin-1 overexpression on doxorubicin suppressed MEK/ERK pathway activation was assessed using western blotting. e Schema illustrates the mechanism by which Syncytin-1 promotes the development of HCC and doxorubicin resistance through activation of the MEK/ERK pathway. Syncytin-1 induced the activation of the MEK/ERK pathway and upregulation of its downstream proteins (such as c-myc, c-fos, c-jun, CCND1, and CDK4) levels in HCC. These genes participated in the malignant progression of HCC by promoting cell proliferation, migration, and invasion. Syncytin-1 also prevented HCC cells from doxorubicin-induced cell apoptosis. DOX, doxorubicin. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 4: Syncytin-1 activates MEK/ERK pathway in HCC.a The upregulated genes in HCC were analyzed by KEGG analysis. b and c The expression of p-MEK1/2 and p-ERK1/2 in 53 pairs of formalin-fixed HCC and adjacent tissues were detected using IHC. Representative images are shown (magnification, ×400). d The expression of p-MEK1/2 and p-ERK1/2 were determined using western blotting. The correlation between the expression of Syncytin-1 and p-MEK1/2 or p-ERK1/2 level was analyzed using Pearson’s correlation test. e The levels of p-MEK1/2, p-ERK1/2, and their downstream proteins (c-myc, c-fos, c-jun, CCND1, and CDK4) were examined in HCC cell lines transfected with Syncytin-1 or pSilencer-shSyncytin-1 by western blotting. f The levels of p-MEK1/2, p-ERK1/2, and their downstream proteins were detected after using MEK/ERK-specific inhibitors (JTP-74057, or GDC-0994) in Huh7 transfected with Syncytin-1. The bars represent results from at least three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig 5: Overexpression of LMP1 activates the MEK1/2/Nrf-2 signaling pathway. The expression of the MEK1/2/Nrf-2 signaling pathway was detected by western blot analysis. All data are displayed in the form of mean ± standard deviation. Two-way ANOVA and Tukey's multiple comparison test are used to test the significant difference of data. *, #p < 0.05, **, ##p < 0.01.