Fig 2: EBV and AFB1 share a common pathway to regulate TGFBI. (a) Schematic representation of the in vitro combined AFB1-exposure and EBV-infection-mediated primary B cells immortalization experiment. (b) qPCR quantification of TGFBI mRNA expression levels in LCL obtained as explained in (a) (* p < 0.05). (c) Heatmap of CG methylation levels in B cells at different stages of EBV-mediated immortalization, in presence or absence of concomitant AFB1 in vitro exposure. Independent experiments from different B cells donors. (d) pyrosequencing-based analysis of TGFBI methylation levels within the three CpGs of interest at different time points of EBV-induced B cells immortalization with concurrent AFB1/DMSO treatment. (e) Immunoblotting quantification of Phospho-IκBα (PIKBA) and IκBα (IKBA) levels in Louckes vs. Louckes-EBV treated or not with AFB1 50 µM. (f) The enzymatic assays-based measure of DNMTs activity (left graph (OD/h/mg)) and DNMT1 quantity in the nucleus (right graph (ng/mg)) in RPMI-LMP1 vs. RPMI-pLXSN cell lines treated with AFB1/DMSO and with Bay11/DMSO.

Fig 3: mTOR hyperactivation upregulates the expression and activity of DNMT1. A Protein levels of DNMT1, S6K, P-S6K, P-4E-BP1 (T37/46), P-4E-BP1 (S65), P-4E-BP1 (T70), and 4E-BP1 determined by Western blot analysis in SNU423 and SNU449 cells stimulated with 100 ng/ml insulin for 12 h or 24 h. The values for P-S6K and P-4E-BP1 were normalized against the band intensities of S6K and 4E-BP1. B DNMT1 activity was detected in SNU423, SNU449 and MHCC-97H cells treated with DMSO and rapamycin (500 nM), as well as in WT and TSC1−/− MEFs, using a DNMT1 assay kit. The y-axis indicates DNMT1 activity as represented by the OD value. Data were presented as mean ± SD and each assay was performed for three times. C Linear correlation between p-mTOR IHC scores and DNMT1 IHC scores in tumoral regions of HCC patients. Correlations were analysed by Spearman’s rank correlation coefficient test. Ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig 4: Inhibition of mTOR suppresses the DNMT1 translation process. A RT–qPCR analysis of DNMT1 mRNA expression in SNU423 and SNU449 cells treated with rapamycin (500 nM) for 24 h. B and C Protein levels of DNMT1 and P-S6K determined by Western blot analysis in SNU423 and SNU449 cells treated with or without rapamycin (500 nM) in the presence of MG-132 (20 μM) (B) or CHX (50 μM) (C) for 12 h. D SNU449 cells were transfected with the modified pGL3 plasmid and treated with rapamycin (500 nM) and vehicle (DMSO). mRNA levels were measured by RT–qPCR, and the luciferase activity was normalized to the transcription level. E Polysome profiles showing the effect of the mTOR signalling inhibitor Torin1 on global translation in SNU449 cells. SNU449 cells were subjected to nutrient deprivation (maintained in 0.1% FBS) for 16 h and were then incubated with nutrient-replete medium (10% FBS) containing Torin1 (250 nM) for 4 h. DMSO was used as a control. F RT–qPCR analysis of mRNA levels in the input lysate from the polysome analysis described in E. G–I The ACTB, RPS20, and DNMT1 mRNA abundances in the fractions from E were quantified by RT–qPCR and calculated as a percentage of the total in all fractions. J Polysome profiles showing global translation in MEFs. K RT–qPCR analysis of mRNA levels in the input lysate from the polysome analysis described in J. L The DNMT1 mRNA abundance in the fractions from J was quantified by RT–qPCR and calculated as a percentage of the total in all fractions. Data were presented as mean ± SD and each assay was performed for three times. Ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig 5: mTOR hyperactivation induces aberrant expression of DNMTs. A Protein levels of DNMTs, S6K, P-S6K, and TSC1 determined by Western blot analysis in WT and TSC1−/− MEFs after treatment with DMSO or rapamycin (500 nM) for 24 h. B Protein levels of DNMTs, S6K and P-S6K determined by Western blot analysis in SNU423 and SNU449 cells subjected to starvation (cultured in complete medium without serum, glucose, and amino acids) for 4 h or 24 h. C Protein levels of DNMTs, S6K and P-S6K determined by Western blot analysis in SNU423 and SNU449 cells starved for 24 h and then recovered by culture in complete medium. D Effect of inhibiting mTOR signalling with rapamycin on DNMT and P-S6K protein expression. Protein levels of DNMTs, S6K and P-S6K determined by Western blot analysis in SNU423 and SNU449 cells treated with rapamycin (500 nM) for the indicated periods. E Protein levels of DNMTs, mTOR, Raptor, and Rictor determined by Western blot analysis in SNU423 and SNU449 cells after siRNA transfection for 48 h. The solid arrows pointed to the correct location of DNMT3B. The values for P-S6K were normalized against the band intensities of S6K. Data were presented as mean ± SD and each assay was performed for three times. Ns, not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001