B1.12: a novel peptide interacting with the extracellular loop of the EBV oncoprotein LMP1

Latent membrane protein 1 (LMP1) encoded by the Epstein-Barr virus (EBV) plays an important role in EBV-induced cell transformation. Down-regulation of the LMP1 expression had shown promising results on cancer cell therapy. In this study, we identified by Phage display a novel peptide called B1.12 (ACPLDLRSPCG) which selectively binds to the extracellular loop (B1) of the LMP1 oncoprotein as demonstrated by molecular docking, NMR and ITC. Using an LMP1 expressing cell line, we showed that B1.12 decreased cell viability, and induced G0/G1 cell cycle arrest. In addition, the expression of A20, pAkt, and pNFkb (pRelA536) in C666-1 cells treated with B1.12 decreased compared to the untreated cells. In conclusion, we selected a novel peptide able to bind specifically to the extracellular loop of LMP1 and thus modulate its oncogenic properties.

Phage display is a powerful technique that allows the presentation of multiple peptides on the surface of filamentous phage particles for diverse applications 1 . Recently, the bio-panning of phage display peptide library on whole cells was proven successful for selecting peptides targeting cancer cells 2,3 . Zhou et al. have identified a tumor cell-binding peptide able to control ovarian cancer cell viability, migration, invasion and adhesion in vitro as well as tumor growth and metastasis in vivo 4 . Furthermore, Ma et al. used a substractive whole cell bio-panning to identify peptides that bind specifically to the membrane of human esophageal squamous cell carcinoma (ESCC). These peptides are very useful for imaging, detection and targeted therapy 5 . In the present study, we focused on the latent membrane protein 1 (LMP1) which is the major oncoprotein of the Epstein Barr Virus (EBV) that is over-expressed in some EBV-associated malignancies such as nasopharyngeal carcinoma (NPC) 6 . Structurally, LMP1 is an integral membrane protein consisting of a short cytoplasmic N-terminus region of 20 amino acids, a trans-membrane domain with six membrane-spanning segments that anchor LMP1 in the plasma membrane, and a long cytoplasmic C-terminal region of 200 amino acids 7 . The cytoplasmic region contains transformation effector sites (TES-1 and TES-2), which are similar to the tumor necrosis factor (TNF) receptor signaling regions and regulate cellular signaling pathways [8][9][10][11][12] . Previous studies have indicated that tumors displaying high levels of LMP1 tend to be more malignant with frequent distant metastasis 8 . Therefore, it is important to reduce the expression level of this oncoprotein to slow tumor progression and invasion, and to improve the response to treatment and thus patient survival. In this context, different approaches had been developed based on the use of DNAzyme, specific antibodies and siRNA [13][14][15][16] . In the present work, we used phage display approach to select peptides targeting the first extracellular loop of LMP1. It is important to mention that to date, there is not a known ligand interacting with LMP1 and that this receptor is activated constitutively 12 . With this in mind, we thought that the search for specific peptides targeting the extracellular LMP1 loops can probably mimic a ligand that could prevent the activation of LMP1 and consequently the signaling cascade controlled by this oncoprotein. Therefore, we synthesized a peptide similar to the sequence of LMP1's first extracellular loop to use it as a target in the bio-panning of the phage library. Usually, the panning is performed using proteins or whole cells: in the present work, we used a synthetic peptide as a target to screen the phage library. After three rounds of panning, we selected 15 phage clones that have binding capacity to the target. Among them 6 were sequenced and we retained Peptide synthesis and analysis of the B1/B1.12 interaction. All peptides: B1 (CMSDWTGG), B1.12 (ACPLDLRSPCG), and B4 (ACNTGSPYECG) were synthesized using standard solid-phase Fmoc chemistry. N-terminus FITC labeled B1.12 was also synthesized. The purity of the peptides was assessed by HPLC to >95% ( Supplementary Fig. S1). By mass spectrometry, the experimental mass-to-charge (m/z) ratios measured for all peptides were as expected (856.9 M, and 1132.2 M, and 1100.41 respectively). These peptides were used to investigate the interaction target/selected peptide using NMR and ITC. As shown on Fig. 2a,b, B1.12 peptide bound specifically to its target B1. Interestingly, on the basis of the NMR spectrum, the interaction between the two peptides can be detected through the chemical shift variation of three amino acids of the B1 peptide which are Met 2 , Trp 5 and Thr 6 residues. Besides, we noticed a conformational variation in the engaged molecule (B1) that is due to the variation in the chemical environment (Fig. 2a). On the other hand, ITC profile confirmed the binding of the B1.12 peptide to its target B1 peptide at 30 °C (Fig. 2b). Aliquots of 2 μL of B1 peptide (0.2 mM) were repeatedly injected into the sample cell containing B1.12 peptide (0.01 mM). As evident from the titration profile, the exothermic heat flow decreases with the increase of the number of injections until saturation was reached. The exothermic binding reaction essentially ceases after nineteen injections when all of the peptide B1.12 molecules present in the cell were bound to the B1 peptide molecules, and the addition of the first peptide causes no more heat flow.

Molecular Docking.
To further validate the binding of the B1.12 peptide to its target, we carried out a docking simulation analysis using the PatchDock and Fire Dock programs. On the basis of global energy amount, we selected the more suitable results. Interaction sites among the interface residues of both the N-terminal region of LMP1 and B1.12 peptide were chased by PSAIA. The model shows that the N-terminal region of LMP1 binds specifically to B1.12 peptide through interactions involving 3 pairs of amino acids (Fig. 3). All involved interactions were either Van der Walls or polar (Table 2). Furthermore, interaction studies on mutants of the ligand affecting the aa required for the peptide-ligand binding as discovered by NMR, have shown that the overall interaction www.nature.com/scientificreports www.nature.com/scientificreports/ energy increases when the Trp is mutated to Ala, attesting the specificity of our selected peptide (WT: −49.82 compared to −45.7 for the mutated model, Table 3). However, by changing the Thr to Ala, the overall energy of the interaction decreases considerably (Table 3). These results suggest that Trp is critical to maintain the binding B1.12/LMP1, but Thr is of less importance for this interaction.     www.nature.com/scientificreports www.nature.com/scientificreports/ Competitive inhibition test for peptide binding. The competitive inhibition test was performed to determine whether the peptide B1.12 and its corresponding phage clone could compete for the same binding site of the target B1 on both HeLa/LMP1 cells as well as the synthetic peptide itself. We demonstrated that with an increase in the concentration of B1.12, the inhibition ratio increased gradually. When the peptide concentration was 400 µM, the inhibition ratio was approximately 43% (Fig. 4a). The unrelated peptide had no significant effect on the binding of positive phage B1.12 to the target B1. Furthermore, competitive inhibition assay was conducted on HeLa cells expressing or not LMP1. Our data showed that the inhibition ratio reached 45% when the peptide concentration is 100 µM (Fig. 4b).
B1.12 affects cell viability of NPC cell line C666.1. To examine whether the peptide B1.12 affects cell viability, we performed MTT assay using C666.1 cell line expressing LMP1. We showed that the viability of the cells gradually decreases with increasing peptide concentration to about 60% in the presence of 200 µM of B1.12. However, the viability of the control cells (HT129) was maintained at 100% in the presence of different concentrations of the peptide B1.12 (Fig. 5).
Effect of B1.12 on cell cycle. It is well known that LMP1 promote cell growth and activate cell cycle progression, therefore, we studied the effect of B1.12 and control peptide on cell cycle progression of the NPC-derived cell line C666.1 and on HT29 cells. After treatment with 50 µM of B1.12, 73% of the cells were blocked at G0/G1 phase compared to 56% for the untreated cells (Fig. 8a,b). Furthermore, when treating with 50 µM of control peptide (Fig. 8c,d), no difference in the C666-1 cell cycle progression was seen compared to the untreated cells (53% www.nature.com/scientificreports www.nature.com/scientificreports/ vs 50.8%). On the other hand, we tested the effect of B1.12 on a control cell line (HT29). Our results showed that the cell cycle phases still unchanged (78.3% vs 75%) with or without B1.12 treatment (Fig. 8e,f).

Effect of B1.12 on LMP1-dependent pathways.
To examine whether the binding of B1.12 to LMP1 affects the LMP1-mediated signaling pathways, we focused on the expression of pAkt and pNFκb (pRelA536) proteins in C666.1 cells treated or not with B1.12 peptide. Western blot showed that the expression of pAkt is low in B1.12-treated-C666.1 compared to those in the untreated or treated with control peptide (Fig. 9a). Furthermore, western blot showed a weak change in the pNFκb (pRelA536) expression with B1.12 in comparison with the untreated C666.1 or treated with control peptide (Fig. 9a). Histograms showing the ratios of pAkt/Tubulin and pRelA536/Tubulin were presented in Fig. 9b. Moreover, we investigated the expression level  www.nature.com/scientificreports www.nature.com/scientificreports/ of A20 which is a well-known LMP1-target in C666.1-B1.12 treated and untreated cells. Interestingly, in the C666.1-B1.12 cells, the expression of A20 decreased compared to untreated cells indicating that B1.12 treatment affected LMP1 signaling pathway (Fig. 9c).

Discussion
NPC is highly prevalent in some regions including southern China and Southeast Asia. EBV infection is constantly associated with NPC as indicated by the presence of viral transcripts and protein antigens in tumor cells 17 . EBV is a lymphotropic human gamma herpes virus that has been implicated in the pathogenesis of several human malignancies including Burkitt and Hodgkin lymphomas, NPC and gastric carcinoma 18 . Among the EBV proteins expressed in NPC cells, LMP1 exhibits properties of a classical oncoprotein. It activates different signaling pathways, such as nuclear factor kappa B (NFκB), c-jun N-terminal kinases (JNK), mitogen-activated protein kinases (MAPK), Janus kinase (JAK), serine-threonine protein kinase (Akt), activators of transcription protein (STAT), and others involved in the proliferation, apoptosis, and metastasis of tumor cells 19,20 . Therefore, LMP1 is considered as the primary oncogene of the EBV and thus a potential target for NPC targeted therapy 21 . It has been shown that down-regulation of LMP1 expression using RNA interference is potentially effective in the prevention of metastatic NPC and the reduction of NPC radio-resistance. Furthermore, Yang et al. designed specific DNAzymes (synthetic, single-stranded DNA catalysts) that can bind and cleave the LMP1 mRNA resulting in suppressing the expression of LMP1 and affecting LMP1-associated signaling pathways 18 . On the other hand, inhibition of LMP1 expression by siRNA in LMP1 positive NPC derived cell line C666-1, lead to an arrest of the cell cycle and increased the sensitivity of the cells to cisplatin 16 .
Phage display technology has been extensively used to identify small molecules, such as antibodies or peptides that bind to specific targets 4,22 . Regarding their properties such as high efficiency in cell-penetrating and strong biding efficacy with low immunogenicity, peptides are good candidates for clinical diagnosis and treatment of various types of cancers 1,2 . Indeed, several works described peptides selected by phage display, and targeting tumor cells. In breast cancer, a αFGF-binding peptide, AP8, isolated by phage display technology, provided an effective αFGF/FGFRs antagonist that might have potential applications in the treatment of breast cancer, characterized by the upregulation of αFGF/FGFRs 23 . Furthermore, Wang et al. identified a novel peptide, (NPMIRRQ), targeting cells and specifically ovarian cancer tissuesthatcan be applied in the diagnosis and treatment of ovarian cancer 24 . A novel peptide GP-5, isolated by whole-cell subtractive panning of the phage displayed 12-mer peptide library, has also been selected and binds specifically to gastric cancer (GC) cells and tissues. This peptide may . Density quantification of each band was assessed by the 'image-j' software. Data are represented as mean ± SD (n = 4 biological replicates), and P-value was evaluated by one-way ANOVA test, ***P = 0.0006, and ****P < 0.0001. Expression level of A20 in both B1.12-treated and untreated C666.1 cells are represented in (c). Data are represented as mean ± SD (n = 3 biological replicates), and P-value was evaluated by two-tailed Mann-Whitney u test (P = 0.1). ns: non significant.
www.nature.com/scientificreports www.nature.com/scientificreports/ thus be used in the molecular imaging for detection and/or in targeted drug delivery, in combination with other biological or chemical agents 25 .
In this work, we used synthetic peptides as target to screen a C-7-C phage display peptide library to identify peptides that specifically bind to the external loop of LMP1. After three rounds of panning, many phage candidates showed strong binding affinity to the target. Among them, the B1.12 phage which bound most effectively to its target as demonstrated by NMR experiments. NMR data showed also that B1.12 peptide interacts with three residues Met 2 , Trp 5 and Thr 6 of the B1 target peptide. Interestingly, almost same residues were involved in the interaction region between the N-terminal domain of LMP1 and the B1.12 peptide investigated by the docking prediction tools.
Further, we used two cell lines namely C666.1 and HeLa/LMP1 to prove the specific binding of the selected B1.12 peptide. For functional studies, we retained C666.1 which is an NPC tumor derived-cell line that maintains LMP1 expression and thus more suitable for testing the inhibitory effect of our peptide. We showed that the cell viability gradually decreased with increasing peptide concentration to about 60% in the presence of 200 µM of B1.12, whereas, the viability of the control cells (HT129) was maintained at about 100% in the presence of different concentrations of the peptide B1.12.
On the other hand, we tested the effect of B1.12 treatment on LMP1-involved signaling pathways. For example, we investigated the expression of pAkt and pNFκb (pRelA536) in C666.1 cells after incubation with the peptide B1.12. We noticed that, the expression of both pAkt and pRelA536 decreased in B1.12-treated-C666.1 compared to those in the untreated or treated with control peptide more markedly for pAkt, suggesting that LMP1 could modulate some signaling pathways through its membrane domain. Indeed, previous studies have shown that LMP1 transmembrane domains interacts to activate NFκB pathway in HEK293 LMP1-transfected cells 26 . In addition, Pratt et al., showed that the involvement of the carboxy-terminal region of LMP1 in the apoptosis regulation of B cells is directly linked to the activity of its six trans-membrane domains 27 .
It is well established that LMP1 induces A20 (TNFAIP3) expression 28 , therefore, we studied the expression level of this gene in C666.1-B1.12 treated and untreated cells. Our data showed that the A20 level decreased in the C666.1 cells after B1.12 treatment confirming that B1.12 modulates LMP1 activity and affects its role in cell signaling pathways especially the NFκB pathway.
In summary, we have identified a novel peptide which specifically binds to the extracellular loop of LMP1 and modulates its oncogenic effect by acting probably as an antagonistic ligand. It is evidently warranted that the B1.12 peptide specifically targets LMP1 expressing cells which provide possibilities for using it in imaging, detection, and targeted therapy. Further studies on animal models holding tumor cells expressing the viral oncoprotein LMP1 are obviously needed to confirm our data.

Methods
Cell lines and culture. Three cell lines were used: C666-1 which is a native EBV-infected NPC-derived cell line constitutively expressing LMP1, HeLa cells transfected with native vector (Gpt) or harboring the cDNA encoding LMP1, and HT-29 which is a human colon cancer cell line All cells were maintained in RPMI medium 1640 supplemented with 5% (V/V) FBS (Gibco), 4 µg/mL Gentamycine and 2 mM L-glutamine at 37 °C in a humidified atmosphere of 5% CO 2 . These cell lines were kindly provided by Pr P. Busson (IGR-France).
Selective Phage enrichment. Peptide harboring phage selection was performed as described below. A solution of 1 mg/mL of the B1 peptide in 100 mM NaHCO 3 (pH 8.6) was prepared and 100 μL was coated on a 96-well plate and incubated overnight at 4 °C. The coating solution was poured off and blocking buffer (skimmed milk at 5 mg/mL in 100 mM NaHCO 3 ) was added for 1 hour at 4 °C. After washing with 0.1% (v/v) Tween-20/TBS, 10 μL (2 × 10 11 plaque forming units, pfu) of original library, Ph.D ™ -C7C Phage Display Peptide (New England Biolabs), were diluted in 100 μL TBST and added to the plate well for 2 hours at room temperature with gentle agitation. After ten washes with 0.1% TBST, the bound phages were eluted with 0.2 M glycine-HCl (pH 2.2) and neutralized with 1 M Tris-HCl (pH 9.1). Eluted phages were amplified in 20 mL LB inoculated with the E. coli ER2738 strain, purified by PEG/NaCl precipitation, and titrated as described in the NEB standard protocol to be used for the next round of selection. The two next rounds of selection were performed under more stringent conditions as the concentration of Tween-20 was gradually increased (0.3% and 0.5% for the second and third round, respectively) and the incubation time of phages with the target was gradually reduced (1.5 hours and 1 hour in the second and third rounds, respectively).
Evaluation by ELISA of the phage-binding to B1 peptide. Fifteen phage clones were picked up randomly from the last round of biopanning, and were individually amplified and titrated. Approximately 1 × 10 10 pfu of phages were added to each well pre-coated with the B1 peptide (100 µg) as previously described and incubated for 1 hour at room temperature and the unbound phages were removed with ten washes with 0.5% TBST. The anti-M13 monoclonal antibody (200 μL of dilution at 1:1000) were added, and incubated at room temperature for 1 hour with gentle agitation followed by incubation with horseradish peroxidase (HRP)-anti mouse for 1 hour. The bound antibody was visualized using 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt ABTS (from Sigma) and the absorbance was measured at OD 410 nm.

Evaluation by ELISA of phage-binding to cell lines. HeLa/Gpt and HeLa/LMP1 (5 × 10 4 cells/per well)
were seeded into 96-well microplates and grown at 37 °C for 24 hours. Subsequently, cells were fixed with 4% paraformaldehyde in PBS and blocked with 5 mg/mL BSA in PBS for 1 hour at room temperature. After washing, 10 10 pfu/well were added and incubated for 1 hour at room temperature with agitation. After washing with 0.5% Scientific RepoRts | (2019) 9:4389 | https://doi.org/10.1038/s41598-019-39732-y www.nature.com/scientificreports www.nature.com/scientificreports/ TBST (10 × 2 minutes) the anti-M13 antibody (GE Healthcare) was added at 1:1000 and incubated for 1 hour at room temperature, followed by washing (6 × 2 minutes) with TBST before adding the anti-mouse antibody conjugated to the peroxydase (Vector Laboratories). The plate was incubated for 1 hour at room temperature, and then washed 6 × 2 minutes with TBST and ABTS (Sigma) was used to detect the bound antibodies as described previously.
DNA Sequencing of selected phages. Positive phage clones giving the strongest ELISA signal were amplified in E. coli ER2738 strain and the M13 single stranded DNA was extracted according to the standard protocol. DNA sequencing was performed using the −96 gIII primer (5′-CCCTC ATAGTTAGCGTAACG-3′).
For ITC, analysis was performed at 30 °C using an isothermal titration calorimeter (VP-ITC, LLC Microcal Inc., Northampton, MA, USA). Peptides were suspended in NaHCO 3 buffer (0.1 M, pH 5.7, after 2 hours of sample equilibration) and the reference cell was filled with the same buffer. B1 peptide (0.2 mM) was placed into 250 μL syringe and 1200 μL of B1.12 peptide (0.01 mM) was placed in the titration cell. The peptide solution was injected into the stirred sample cell containing B1.12 peptide in >20 injections (2 μL per injection) by a computer-controlled pump. The injection lasted 10 seconds with an interval of 120 seconds between successive injections. Heat released with each injection was measured (Differential Potential in µCal/sec) by the calorimeter in dynamic correction mode. Control experiments were performed by tittering the B1 peptide into the buffer solution at the same pH and concentrations with and without the B1 peptide, in order to discriminate the interaction process against the dilution of B1 and its possible self-association, respectively.

Molecular docking.
We constructed a three-dimensional model structure of the N-terminal region of the LMP1 protein based on the structure of the sweet transporter 29 (PDB: 5ctgA), the Anabaena sensory rhodopsin 30 (PDB: 1XIO), the NE. Coli YajR transporter 31 (PDB: 3WDO) and the membrane domain of respiratory protein of E. coli 32 , (PDB:3RKO), by the use of GPCR-I-Tasser 33 . This module is a hybrid method designed for 3D prediction of trans-membrane proteins. The GPCR-I-Tasser pipline includes three steps, the first one consists of the generation of the trans-membrane helix, the second one consists of the I-Tasser fragment structure reassembly simulations and the third one consists of the selection of the model and the atomic refinement. Iterative Threading ASSEmbly Refinement server (I-TASSERcoupled to the GPCR) (http://zhanglab.ccmb. med.umich.edu/GPCR-I-TASSER/) template structures were selected by multiple threading approach Local Meta-Threading-Server (LOMETS) based on sequence similarity to the structure described at the top (Sweet transporter protein, the anabaena rhodopsin….). Alignments of the query sequence with each template were generated using the ConSurf server. This program provides multiple sequence alignment with conserved features, which provides information about specific amino acid features in their local environment. A total of five three-dimensional structure models were generated by GPCR-I-TASSER, among them, the best model was identified based on confidence score (C-Score) and the refined model coordinates were evaluated by MolProbity 34 . As the LMP1 atomic level structural data was not yet available, we constructed a model as described in our previous work 35 , and the B1.12 peptide model was generated similarly (all models are available as Supplementary Materials). To obtain a better understanding of the interaction between the N-terminal domain of LMP1 and the B1.12 peptide, we performed the modeling of the peptides binding to LMP1 in order to predict the positions and the orientation of the peptides in her binding cleft. We used a rigid protein-protein docking software packages that are based on different approaches, namely PatchDock 36 and FireDock 37 . The PatchDock algorithm is inspired by object recognition and an image segmentation technique used in computer vision and applies geometric hashing and poseclustering matching to match concave and convex patches of interacting surfaces. A refined model of the complex using the NMR constraints was generated by patchDock. The web server is located at http://bio-info3d.cs.tau.ac.il/FireDock/. FireDock transforms the model candidates generated by PatchDoc, and performs the refinement using a restricted interface side-chain optimization to allow a certain amount of steric clashes. The refined candidates are scored and ranked according to the energy function. A Final step of refinement was assessed with FG-MD 38 , which is a molecular dynamics algorithm for atomic-level protein structure refinement. FG-MD aims to refine the initial models closer to the native structure. It can also improve the local geometry of the structures by removing the steric clashes and improving the torsion angle and the hydrogen-binding networks. A final analysis with MolProbity was assessed to validate and to analyze the quality of the structures. Moreover, to better confirm the B1.12-LMP1 interaction, we have established two mutant models that are affected in the essential aa required for the interaction as indicated by NMR. As previously described, the Firedock software was used and the interaction of the different LMP1 mutants with the B1.12 peptide was studied.
Competitive inhibition. Binding of the B1.12 peptide to its target B1 and to HeLa/LMP1 cells was evaluated by a competitive binding assay. For competition on B1 as a target, the B1.12 peptide or the unrelated control peptide were incubated at concentrations of 1, 5, 10, 25, 50 and 100 µM with B1 peptide (100 µl of a solution of 1 mg/ mL in NaHCO 3 ) at room temperature for 1 hour. Then the B1.12 phage (2 × 10 10 pfu) was added to each well and incubated for 1 hour. After washing, the bound phage was detected by the anti-M13 antibody as described previously. For the competition test on HeLa cells expressing or not the viral oncoprotein LMP1, cells were seeded into 96-well plates (10 4 cells/well), washed twice in PBS, and fixed with 4% paraformaldehyde for 30 minutes at room temperature. Synthetic peptides at concentrations of 50, 75, 100 and 200 µM were incubated with the cells at 37 °C for 1 hour and the experiment was carried out as described above. All experiments were done in triplicate and the mean values of OD were used to calculate the rate of competitive inhibition according to the following formula: inhibition ratio = (OD control − OD phage)/OD control × 100, where OD control and OD phage represent the OD450 nm values of phage binding on cells incubated with PBS and with the peptide, respectively.
Cell viability assay. Cells were plated into 96-well plates until 80% confluent and then treated with different concentrations of B1.12 peptide (100, 200, 300 and 400 µM). After 48 hours of incubation, 20 μL MTT (5 mg/mL) were added to each well. After 4 hours, 100 μL dimethyl sulfoxide (DMSO) was added to each well, and absorbance was measured at 570 nm using a Varioskan ™ Flash Multimode Reader (Thermofisher Scientific). The percentage of cell viability was calculated using this formula:(OD590 nm of treated cells/OD590 nm of untreated cells) × 100.
In vitro cellular uptake by flow cytometry. The cellular uptake of the FITC labeled B1.12 peptide/ unrelated peptide was analyzed by flow cytometry. C666.1, HeLa/LMP1, and HeLa/Gpt cells, were seeded at a density of 4 × 10 5 cells/mL at 37 °C for 24 hours, and then incubated with 100 μM of both FITC-labeled peptides for 24 hours. Cells were washed three times, trypsinized, centrifuged at 1700 rpm for 5 minutes and then resuspended in 0.35 mL of PBS. The fluorescence associated to the cells was measured using LSR Fortessa X-20 (Becton Dickinson) for HeLa/LMP1 or Attune NxT Flow Cytometer (Thermofisher) cytometer for C666.1 and HeLa/Gpt cells.

Fluorescence microscopy.
To further investigate the fixation of the peptide on C666.1 cells, fluorescence microscopy was performed. Approximately 1 × 10 6 cells were washed 3 times with PBS, fixed with 4% parafolmaldehyde for 15 minutes, blocked by 5 mg/mL BSA for 1 hour, and incubated with 10 μM FITC labeled B1.12 peptide or anti-LMP1 antibody (1/500 Dako) for 1 hour at room temperature. After being washed with PBS, the fixation of LMP1 was detected by Alexa-fluor 405 labeled secondary antibody (1/100). HeLa/Gpt cells were also stained as described above to serve as negative control. Images were analyzed using an EVOS Floid Cell Imaging Station (Life Technologies).
Cell cycle analysis. Cell cycle analysis of C666.1 and HT29 cells was performed using the FXcycleTm PIi/RNnase staining solution (Invitrogen). Briefly, cells (4 × 10 5 ) were seeded and grown for 24 hours, and then incubated with serum-free RPMI for another 24 hours (starvation) to decrease cell constitutive signaling. After starvation, cells were incubated or not with 50 µM B1.12 peptide (C666.1 and HT29 cells) or control peptide (C666.1 cells) for 48 hours. Following incubation, cells were washed with PBS, trypsinized and centrifuged at 1200 rpm for 5 minutes. The cell pellet was then washed twice with PBS, and cells were fixed with 5 mL of ethanol 70% for 24 hours at 4 °C. Aliquots of 2 × 10 5 ethanol-fixed cells were then centrifuged for 5 minutes at 450 g, washed with PBS, and then resuspended in Propidium Iodide Reagent and incubated for 30 minutes at room temperature, shielded from light. The data were collected and analyzed by an Attune NxT Flow Cytometer (Thermofisher).
Protein extraction and western blot. Cells were cultured at 80% confluency and then incubated with 100 µM B1.12 peptide for 24 hours. Subsequently, cells were washed twice with cold PBS and scrapped in ice-cold RIPA buffer (50 mM Tris pH 7.4, 5 mM EDTA pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5% NaDOC, 0.5% NP 40) supplemented with 5 mM PMSF. After 30 minutes on ice, cells were sonicated, and cell debris were removed by centrifugation at 15,000 rpm at 4 °C for 15 minutes. Proteins were quantified using the Bradford Protein Assay (Bio-Rad) according to the manufacturer's specifications. Thirty μg of total proteins were separated on 10% SDS-PAGE and transferred to PVDF membrane (Amersham Hybond-P). The membranes were blocked at 4 °C overnight with PBS 0.1% Tween 20 containing 5 mg/mL skimmed milk, prior to be incubated with pAkt (Biotechne, diluted 1/2500) pRelA536 (Biotechne, diluted 1/1000), and Tubulin (Thermofisher, diluted 1/1000) antibodies for 2 hours at room temperature, followed by incubation with anti-rabbit or anti-mouse conjugated to horseradish peroxidase at room temperature for 1 hour. The target protein band was visualized using the enhanced chemiluminescence detection system (Amersham ECL Prime Western Blotting Detection Reagent) and luminescent bands were visualized using the VERSADOC imaging system. Band densities were assessed by Image-J software.
www.nature.com/scientificreports www.nature.com/scientificreports/ cDNA synthesis and RT-qPCR analysis. Total RNA from C666.1 cells treated or not with B1.12 and control peptides was extracted using TRIzol reagent. cDNA was synthetized using the reverse transcription kit (Invitrogen), and served as a template for detecting A20 gene expression by Real-time quantitative PCR using SYBR Premix Ex Taq (Tli RNase H Plus, Takara) and using CFX96 (Biorad). The housekeeping gene GAPDH was used as an internal control to normalize the expression levels of the gene. The primers used for the amplification of the A20 gene are as follows: forward: 5′-GCCCGAAGTGGACTTCAGTACAAC-3′, and reverse: 5′-GGCGAAATTGGAACCTGA TTCCAAACTT-3′. Relative quantification of mRNA expression was performed by the comparative 2−ΔΔct method to calculate the difference between amplifications of A20 and GAPDH. The experiments were performed with three technical replicates, and the mean fold changes and standard deviation were also calculated. Statistical analysis. Statistical analysis was performed by GraphPad ™ Prism 5.0 software (Graphpad Software Inc., San Diego, CA, USA) and values were considered significant when P < 0.05.

Data Availability
The data that support the finding of this study are available from the corresponding author upon request.