A new p65 isoform that bind the glucocorticoid hormone and is expressed in inflammation liver diseases and COVID-19

Inflammation is a physiological process whose deregulation causes some diseases including cancer. Nuclear Factor kB (NF-kB) is a family of ubiquitous and inducible transcription factors, in which the p65/p50 heterodimer is the most abundant complex, that play critical roles mainly in inflammation. Glucocorticoid Receptor (GR) is a ligand-activated transcription factor and acts as an anti-inflammatory agent and immunosuppressant. Thus, NF-kB and GR are physiological antagonists in the inflammation process. Here we show that in mice and humans there is a spliced variant of p65, named p65 iso5, which binds the corticosteroid hormone dexamethasone amplifying the effect of the glucocorticoid receptor and is expressed in the liver of patients with hepatic cirrhosis and hepatocellular carcinoma (HCC). Furthermore, we have quantified the gene expression level of p65 and p65 iso5 in the PBMC of patients affected by SARS-CoV-2 disease. The results showed that in these patients the p65 and p65 iso5 mRNA levels are higher than in healthy subjects. The ability of p65 iso5 to bind dexamethasone and the regulation of the glucocorticoid (GC) response in the opposite way of the wild type improves our knowledge and understanding of the anti-inflammatory response and identifies it as a new therapeutic target to control inflammation and related diseases.


Scientific Reports
| (2021) 11:22913 | https://doi.org/10.1038/s41598-021-02119-z www.nature.com/scientificreports/ to the nucleus to control gene expression 7 . The dysregulation of NF-kB activity is linked to autoimmune and metabolic diseases, inflammatory disorders and cancer 8 . NF-kB plays a pivotal role in the activation of the immune response, modulates cell proliferation and differentiation as well as cell death 9 . Coronavirus disease 2019  caused by Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) has emerged in China and drastically spread throughout the world quickly and declared by World Health Organization as pandemic. Clinical manifestations of COVID-19 may differ from person to person, primarily are characterized by Acute Respiratory Distress Syndrome (ARDS) but an elevated level of pro-inflammatory molecules has been reported as the key pathogenic hallmark of COVID-19 10,11 . It is also very well known the involvement of NF-kB pathway in the regulation and in the pathogenesis of inflammatory diseases 12,13 . NF-kB and Glucocorticoid Receptor (GR) respectively mediate pro-and anti-inflammatory physiological balance. GR is a ligand-activated transcription factor belonging to the superfamily of nuclear receptors. After binding glucocorticoids, GR translocates to the nucleus regulating target genes expression by recognizing Glucocorticoid Responsive Element (GRE) in specific promoters and acts as anti-inflammatory agent and immunosuppressant 14,15 . GR represses NF-kB activation blocking its access to DNA binding sites (-kB) by a protein-protein interaction. It also induces the synthesis of the NF-kB inhibitor IkB 16 . Thus, NF-kB and GR are physiological antagonists in the inflammation process 17 . It has been shown that NF-kB pathway also plays a major role in the development of liver diseases correlated with inflammation like fibrosis and hepatocellular carcinoma (HCC) [18][19][20] . Alternative splicing is a physiological process in eukaryotes and allows to produce different mRNAs from a single gene. Splicing is often tightly regulated in a tissue specific manner producing diverse mRNAs encoding multiple proteins with distinct biological effects [21][22][23] .
In this report we describe and characterize a new alternative splicing form of p65, named p65 isoform 5 (p65 iso5) related to human product, that is functionally distinct from the known isoforms of p65.

Results
Genomic organization of p65 iso5. These investigations started by screening of a mouse cDNA library for the study of differentially expressed p65 binding proteins. Here, a clone with a different relA 5′ end gene arrangement has been isolated. In order to verify if this gene corresponded to an alternative spliced form a series of reverse transcriptase and polymerase chain reactions (RT-PCR) were performed on RNA extracts from mouse and human tissues. Using specific oligonucleotides for the exon 2 and oligonucleotides located upstream of the 5′ untranslated region (5′ UTR) of the p65 gene we found an alternative spliced form of p65 mRNA. This new isoform of p65 was named p65 iso5 (Deposited sequence: GenBank accession number MN508965) and contained a previously unknown exon, (named exon − 1) located upstream to the first known exon of p65 (exon 0) (Fig. 1a). A 5′-RACE mapping performed with mouse brain RNA shown that the exon − 1 has a length of 498 base pair (bp) and has a strong homology (73%) with the 213 bp of the exon − 1 that was cloned from human brain RNA (Fig. 1b). The homology between amino acid sequences of human and mouse isoforms is shown (Fig. 1c).
Because two splice variants of p65 are already known in mouse, if referred to this organism, this new gene product is called p65 isoform 3 (p65 iso3) (Deposited sequence: GenBank accession number MN508964). p65 iso5 spliced the exon − 1 and exon 1 and skipped exon 0. Consensus intronic sequences at splice junctions were present in the murine and human exon − 1 (Fig. 1d). The exon − 1 for p65 iso5 is transcribed in the same direction of the entire relA gene both in mouse and in human and it is a non-coding region. These evidences support the presence of a different promoter region upstream of the previously known. The p65 iso5 ORF (Open Reading Frame) starts at methionine 32 present in the exon 2 ( Fig. 1a). This ATG is bordered by a Kozak consensus-like sequence in both the human and the mouse transcripts (Fig. 1e). Consequently, the resulting p65 iso5 protein lacked the amino acid residues 1-31 and had a shorter RHD that contains the dimerization, nuclear-localization and DNA-binding domains (Fig. 1f).
Identification of p65 iso5 mRNA in human and mouse tissues and the capacity of its protein product to bind kb consensus. Using nested PCR with primers specific for exon − 1 and exon 5-10, the entire transcripts containing the − 1 exon were found both in mouse and human brains RNA (Fig. 2a). p65 iso5 appears as an alternative splice variant of the p65 gene and no transcript containing both the − 1 and the 0 exons were found when PCR was performed with a set of specific oligos for exons − 1/2 and no transcript were observed using specific oligos for exons − 1/0 (Fig. 2a). Entire p65 iso5 mRNA was present in all the nine mouse tissues we analyzed by nested PCR (Fig. 2b) and in human liver or peripheral blood mononuclear cells (PBMC). Both p65 isoforms were able to bind NF-kB consensus DNA as shown by an Electro Mobility Shift Assay (EMSA). For this experiment, expression plasmids encoding p65 iso5 and p65 wild type (wt) were in vitro transcribed and translated and the final product used for EMSA, using a radiolabelled oligonucleotide containing the NF-kB binding motif (Fig. 3a,b). The p65 iso5 mRNA was detected in every tissue we examined and both p65 proteins are able to bind to DNA.
Transcriptional activity of p65 iso5 on NF-kB and GRE promoters and cell localization. Despite p65 iso5 is able to bind to DNA, it activates transcription trough canonical NF-kB responsive elements ten time less than p65 (Fig. 3c). These results indicate that in addition to the two already known trans-activating domains mapped within the COOH terminal region of p65 24 , the small portion deleted in the NH 2 terminal region of the RHD domain of p65 iso5 is also involved in conferring transcriptional activity to p65. NF-kB and GR are oppositely regulated and repress each other transcriptional activity 25,26 . Considering that the physical interaction between p65 and GR has been reported, the domains required for a direct interaction between the two proteins are also present in the p65 iso5 protein 27 . Administration of the selective GR agonist dexamethasone activated the GRE-dependent reporter as expected and this effect was largely reduced by p65. Strikingly, cotransfection with p65 iso5 had exactly the opposite effect doubling dexamethasone-induced gene transcription (Fig. 3d) Fig. S1). Moreover, through immunofluorescence analysis, we demonstrate a different localization of p65 iso5 and p65. The p65 iso5 is primarily localized in the nucleus and in the perinuclear region while p65 is distributed in the cytoplasm (Fig. 3e,f).
Transcriptional activity of p65 iso5 on human interleukin 6 and TNFα promoters. Because it is common knowledge that NF-kB is a dimer among the members of Rel structural-related proteins that regulates several genes involved in inflammation, we analyzed the p65 iso5 transcriptional activity with either p50 or p65 wt on human interleukin 6 (IL-6) or tumor necrosis factor-α (TNFα) promoters. IL-6 is a cytokine that is produced during inflammation playing a key role in the acute phase response process. In addition, IL-6 can also drive chronic inflammation 28,29 . To investigate whether p65 iso5 may have an influence on IL-6 expression, we transfected HeLa cells with pIL6-luc651 promoter construct 30 with heterodimers combination of the three members p50, p65, p65 iso5. As shown in Fig. 4a the p65 iso5/p50 and the p65 iso5/p65 activate transcription  www.nature.com/scientificreports/ more efficiently than the classical p65/p50 dimer. Mutation in the consensus of AP-1, or CRE, or C/EBP binding sites did not significantly affect the transactivation activity of every dimer except for the classical p65/p50 dimers on AP-1 mutated consensus binding sites (Fig. 4a). Surprisingly, despite point mutation of the NF-kB consensus sequence abolished the activation in both dimers containing the p65 wt, the trans-activating capacity of p65 iso5/p50 was unaffected at all suggesting a different way to regulate this promoter region. A clear reduction of trans-activating activity of the dimer containing the p65 iso5/p50 was detected in the mutant on the GRE sequence (Fig. 4a). TNFα is a multifunctional cytokine produced by several types of cells in particular cells of the monocytic lineage 31,32 playing a key role in the regulation of immune cells. Therefore, we studied the possible effects of p65 iso5 protein with either p50 or p65 on human TNFα promoter with or without dexamethasone. The results demonstrate that the heterodimer p65 iso5/p50 has a greater trans-activating activity compared to the classic heterodimer p65/p50 in presence of dexamethasone 33 (Fig. 4b). The data obtained on IL-6 and TNFα promoters shows a different regulation capacity of p65 iso5 compared to p65 wt. The ability of this new isoform to activate the promoters of some target genes, in a different way compared to wild type, suggests that p65 iso5 could help providing different biochemical properties to NF-kB depending on the partners involved in the complex.
Hypothetical binding of p65 iso5 and dexamethasone. Since a possible reason of the ability of p65 iso5 to doubling dexamethasone effect could be determined by a direct binding of p65 iso5 with dexamethasone, we have performed in silico docking simulations in order to investigate the potential of the dexamethasone to bind the p65 iso5 protein. Since the crystallized structure of the complex IkBα/NF-kB was available (PDB: 1NFI) and the p65 iso5 differs from the wt by the mere lack of the first 31 residues, a protein structure model of the p65 iso5 was created with Modeller v9.8 [34][35][36] as shown in on Fig. 5a,b. In addition, the Autodock v4.2 program 37 was used to understand if also p65 has the potential to bind the dexamethasone ( Supplementary Fig. S2). However, the simulations did not find any docking clusters on the p65 wt. The docking analysis between the p65 iso5 model and the dexamethasone revealed four different docking clusters and the top scoring docked model was selected ( Supplementary Fig. S2). The model was characterized by a binding energy of ΔG (kcal/mol) = − 9.09 a value comparable to the one of the binding dexamethasone-human GR ΔG (kcal/mol) = − 11.57 (Supplementary Movie S1). The docking simulation suggests that dexamethasone targets a binding pocket that was occupied by the first 31 amino acids in the wt protein (Fig. 5c). These 31 aa, that are missing in the p65 iso5 ( Fig. 5d), create a β-sheet element that does not allow the binding of the dexamethasone in the wt structure (Supplementary Movie S2).
In vitro binding interaction of p65 iso5 protein with dexamethasone. In order to have an experimental demonstration of the interaction between p65 iso5 and dexamethasone, the Bioluminescence Resonance Energy Transfer (BRET) technique 38 has been performed. We used the Renilla luciferase (Rluc) as the donor and Dexamethasone Fluorescein (Dex-FITC) as the acceptor. We prepared the full-length GR, p65 and p65 iso5 constructs with Rluc fused in frame with the C-terminal regions (Fig. 5e). We initially examined the GR-Rluc/Dex- We used the p65-Rluc construct as negative control and no differences of raw BRET signal ratio were observed with and without Dex-FITC. Finally, an increase of raw BRET ratio, similar to GR-Rluc/Dex-FITC, was observed when p65 iso5-Rluc construct was used. These results confirm that p65 iso5 bind the dexamethasone. So far, GR is the only known receptor able to mediate actions of glucocorticoids on gene transcription and regulation. The capacity of p65 iso5 to bind dexamethasone and the regulation of the glucocorticoid responses in the opposite way of the wild type, opens a new paradigm that needs to be clarified with regard to human inflammation-related diseases.
Evaluation of the mRNA expression levels of p65 and p65 iso5 in PBMC from COVID-19 patients. As it has been pointed out in the study conducted by Huang et al. 39 the levels of TNF-α, IL-2, IL-10, IL-7, and other inflammatory markers were higher in patients suffering from COVID-19 than in healthy subjects. Moreover, it has been observed that the most severe cases of COVID-19 are associated with a reduction in CD4 + T, T-lymphocytes, CD8 + T cells and an increase in levels of C-reactive protein, D-dimer, ferritin, IL-6, IL-2R 40 . Moreover, the transcription factor NF-kB regulates the activity of the immune cells and is involved in the regulation of cytokines gene expression. Taking this into account, an abnormal activation of the NF-kB activ- Taking into consideration the data obtained on IL-6 and TNFα promoters, in which p65 iso5 shows a different regulation compared with p65 wt, we decided to study gene expression profiles by qPCR, for p65 and p65 iso5 in patients affected by SARS-CoV-2 infection. As shown in Fig. 6a, we found that the expression levels of p65 and p65 iso5 mRNA are up-regulated in PBMCs from COVID-19 patients. Furthermore, as shown in Fig. 6b, the blood IL-6 levels in patients with SARS-CoV-2 are higher than healthy patients. Considering that the heterodimer of p65 iso5 with either p50 or p65 amplify the transcriptional activity of IL-6 and TNF-α promoters and the p65 iso5 mRNA levels are higher in COVID-19 subjects, p65 iso5 may be involved in the onset of cytokine storm and development of severe symptoms in patients with COVID-19. These results confirm the involvement of NF-kB in disease progression 41 .
Identification of p65 iso5 protein in human liver tissue samples. In tumor cells, several molecular alterations could compromise the activation mechanism of NF-kB leading to genes deregulation involved in the control of cell cycle, apoptosis, cell migration or adhesion. Since alteration in some of these processes can determine cancer progression, it is clear that there is a connection between NF-kB and carcinogenesis 42,43 . Because www.nature.com/scientificreports/ NF-kB is involved in the regulation of inflammatory response, we analyzed the p65 protein profile in inflammation liver disease by coupling two-dimensional polyacrilacrylamide gel electrophoresis (2D-PAGE) 44,45 and western blotting. As control, the expression profile of p65 iso5 protein was analyzed in transfected Cos-1 and HeLa cell lines with two different antibodies specific for the NH 2 and COOH epitopes (Fig. 7a). Subsequently, we investigated the expression of p65 iso5 in the hepatocarcinoma cell lines HepG2 and HUH7 (Fig. 7b) and in liver extracts isolated from cirrhosis and HCC patients using healthy condition as control for the identification of p65 and p65 iso5 products (Fig. 7c). A more complex isoelectrofocusing pattern for p65 isoforms was shown in HCC and cirrhosis patients in comparison with control condition. The 2D-PAGE analysis, based on predicted isoelectric point (pI) and molecular weight (Mw), shows that these patterns (Fig. 7d) are compatible with the presence of the p65 iso5 and p65 protein in pathological samples, while only p65 is detected in healthy control samples (Fig. 7c). The functional characteristics of the protein encoded by this spliced mRNA are very different from the ones of the canonical p65. Such as the p65 wt, p65 iso5 mRNA is expressed in every mouse and human tissue  www.nature.com/scientificreports/ tested but whilst the p65 wt protein is constitutive expressed in the liver 46 , p65 iso5 gene product is detected only under specific conditions associated with inflammation-related liver diseases. This is an additional different feature of p65 iso5 compared to p65 wt, that adds a further level of complexity to the regulation of NF-kB pathway.

Discussion
Under physiological conditions, activation and resolution of inflammatory responses are regulated by a cascade of cellular events promoting the repair and healing. It is very well known that chronic inflammation can cause tissue damage and pathology in the organism as the development and progression of human cancers 47 . It is known that there is a cross talk between NF-kB and GR pathways and this is involved in pro-and anti-inflammatory response. NF-kB induces the transcriptional activation of genes involved in the stimulation of immune and inflammatory response. The GR, on the contrary, represses NF-kB and induces the expression of anti-inflammatory genes.
Here we showed that exist a new splice variant of p65, named p65 iso5, that is transcribed starting by a new identified exon located upstream of the first known exon. This is the only p65 variant present both in human and mouse showing the importance of this gene product throughout evolution. This new isoform, in the presence of synthetic GC, enhances the GR-mediated anti-inflammatory response. Moreover, this protein, according to the partner with which it forms the dimer, is able to activate the promoter of some target genes in a different way compared to wild type. As demonstrated by docking simulation and BRET assay results, p65 iso5 is able to bind dexamethasone, showing a very different capacity in regulating the effects mediated by GCs. Our results strongly suggest that this isoform can contribute in a fine and more complex way to the inflammatory response.
Since the anti-inflammatory response of GR is also mediated by transcriptional activation of its target genes 48 , the p65 iso5 interaction with dexamethasone could amplify and improve the GR-mediated anti-inflammatory response. Our results are consistent with the idea that the p65 iso5 expression is present in every tissue and cell line analyzed. The expression and the physical interaction between p65 iso5 and GR are critical for target genes transcriptional control. We hypothesize that this complex could control the GR's anti-inflammatory actions in a locus specific manner. The study of the physical interaction between p65 iso5 and GR could highlight the importance of glucocorticoids to control the inflammatory response both in physiologic or pathological conditions. The discovery of a new p65 isoform with different biochemical property, compared with the wild type, and its different ability to activate a broad range of target genes can shed some light on the fine-tuning of inflammation resolution. In fact, we believe that the distinct features of p65 and p65 iso5 could provide a new ground to explain the complex and sometime opposite pro-and anti-inflammatory roles functions attributed to NF-kB 49 .
In some studies, it has been observed that both the nucleocapsid protein and the spike protein of SARS-CoV were shown to induce pro-inflammatory cytokines via activation of the NF-kB pathway 50,51 and that this has a key role in the SARS-CoV-2 infection 52,53 . Here we demonstrate that, p65 iso5 is overexpressed in the PBMC of COVID-19 patients and, in our knowledge, this is the first example showing a new gene product associated with the pathological profile of Sars-CoV-2 infection. Considering the ability of the new isoform p65 iso5 to bind dexamethasone and to regulate the glucocorticoid responses in the opposite way of the wild type, p65 iso5 could play a therapeutic role in alleviating the severe form of COVID-19. In fact, the role of steroids in these patients is to inhibit the expression of certain molecules involved in pneumonia associated inflammatory response 54 . We propose a model in which p65 iso5, according to the homo and heterodimeric complex, can be considered as a stronger pro-inflammatory response. Indeed, this new isoform can dimerize with either p65 or p50 to coordinate an onset phase of different mediators. In addition, p65 iso5 could interact with GR through the formation of a complex and, after treatment with glucocorticoids, amplify the transcription of its target genes involved in the anti-inflammatory response ( Supplementary Fig. S3). We also show that p65 iso5 protein expression is associated with inflammation-related liver diseases. In fact, the p65 iso5 protein is present only in the liver samples of patients suffering from cirrhosis and HCC. This result strongly suggests a role for p65 iso5 to contributing to the onset of these inflammatory liver diseases identifying this new protein as potential new therapeutic target for specific GC ligands that could improve clinical and long-term treatments with GCs.

Methods
Animal and human samples. All animal procedures were conducted following the ARRIVE (Animals

Isolation of peripheral blood mononuclear cells. Peripheral Blood Mononuclear Cells (PBMC) have
been isolated from venous blood samples of 21 patients affected by SARS-CoV-2 and 21 healthy subjects, for a total of 42. PBMC were isolated from whole blood through density-gradient centrifugation, by using Ficoll Paque (GE Healthcare), following manufacturer's instructions.

RNA extraction and RT-PCR.
Mouse or human total RNA was extracted using the RNA NOW reagent (Ozyme) or TRIzol™ Reagent (Invitrogen™). All the experiments were performed in triplicate. The RNA (1 or 2 μg) was reverse transcribed with the using Oligo (dT) primers, RNAsin RNase Inhibitor, M-MLV Reverse Transcriptase and dNTP from Promega. After extraction the RNA was quantified with a spectrophotometer and electrophoresed on agarose/formaldehyde gel to control the quality of the samples. A dilution (1:20) of the RT reaction was used for PCR reaction with specific primers for exon -1/exon 2, exon − 1/exon 0, exon 0/exon 2 (forward exon − 1 primer 5′-GGC CTG GGC CTC TCC CTG CGC AGG GCG AAT G-3′; forward exon 0 primer 5′-CTT TAG CGC GCC GTG GGC TCA GCT GCGA-3′; reverse exon 2 primer 5′-ATG GTG GGG TGT GTC TTG GTG GTA TCTG-3′; reverse exon 0 5′-TCC CGG GGG CGG GGC CGG GGT CGC AGCT-3′). One-fifth of the reaction was analyzed on agarose (2%) gels. Nested PCR (a 1:200 dilution of the first PCR reaction) was used to specifically amplify the entire p65 iso5 mRNA product from heart, brain, spleen, lungs, liver, kidneys, skeletal muscle, testicles, thymus of mouse and brain, liver, peripheral blood mononuclear cell (PBMC) of human RNA. In the first reaction, for the mouse sample, specific primers for exon − 1/3′UTR (untranslated region) were used (forward exon − 1 primer 5′-GGC CTG GGC CTC TCC CTG CGC AGG GCG AAT G-3′; reverse 3′UTR primer 5′-AGG CTT CAG TGC CCT GAA ACC TGG TG-3′). One tenth of the first PCR product was diluted in 250 μl of distilled water and 5 μl were used for the second reaction of the nested PCR exon − 1/exon 0, exon In vitro transcription/translation and electrophoretic mobility shift assay (EMSA) of p65 and p65 iso5. The mouse p65 iso5 with 169 bp of exon − 1 and the p65 cDNA were cloned into pBluescript vector (Stratagene). Uncut plasmid DNAs were transcribed and translated using TNT kit (Promega) following manufacturer's instructions. The in vitro unlabelled translated proteins were incubated with a double-stranded 32 P-labelled oligonucleotide probe containing the specific recognition sequence for NF-kB (5′-AGT TGA GGG GAC TTT CCC AGGC-3′), and analyzed on non-denaturing 5% acrylamide gel. Additionally, to the labelled NF-kB probe an unlabelled oligonucleotide was added in excess (100:1) when appropriate.
Luciferase assay. 24  www.nature.com/scientificreports/ with 25 ng of the phRL-TK (Promega) vector encoding the Renilla luciferase as internal control. For the experiment with the pNFkB-luc reporter, cells were cotransfected with plasmid encoding either the human p65 or p65 iso5 cDNA (250 ng) under the SV40 promoter. The enzymatic activity was measured 24 h after transfection. For the experiment with the pGRE-Luc reporter, cells were cotransfected with either p65 or p65 iso5 plasmids (500 ng). The day after transfection, the cells were treated with 5 × 10 -8 M of the GR agonist Dexamethasone (Sigma) in supplemented fetal bovine serum medium treated with Charcoal-dextran (Hyclone) and the enzymatic activity was measured 24 h later. For both the pNFkB-luc and pGRE-luc experiments the total amount of DNA (1.5 μg) for each transfection was kept constant by adding the appropriate quantity of empty plasmid. The enzymatic activity of the two individual reporter enzymes (Firefly and Renilla) was measured in 20 μl of cell lysate with the Dual-Luciferase Reporter Assay System (Promega) according to manufacturer's instruction using a luminometer LUMAT LB 9507 (Berthold Technologies). All the experiments were performed in triplicates and replicated twice.
In silico interaction and docking studies of p65 iso5 and dexamethasone. The amino acid sequence of our p65 iso5 was used to perform a BLAST research in order to recover a PDB structure of a template. The amino acid sequence of the human p65 iso5 was obtained starting from the amino acid 32 of sequence of p65 wt UniProtKB -Q04206 (https:// www. unipr ot. org/ unipr ot/ Q04206). The 3D structure of p65 iso5 was built with Modeller v9.8 35  www.nature.com/scientificreports/ Tween 20 0,1%, with 100 μl/l of protease inhibitor cocktail) at room temperature and incubated for 2 h in a milk solution, with NF-kB p65 (G-8) (sc-398442) or NF-kB p65 (c-20) (sc-372) antibody dilution 1:5000 or NF-kB p65 (A) antibody (sc-109) (Santa Cruz Biotechnology) dilution 1:5000. Then the membranes were washed 2 times for 7′ with PBS 1×, Tween 20 0,1% and incubated for 1 h at room temperature in a milk solution containing anti-rabbit IgG-HRP secondary antibody (sc-2004 Santa Cruz) dilution 1:4000 when used NF-kB p65 (A) or NF-kB p65 (c-20) antibody and with anti-mouse m-IgGκ BP-HRP secondary antibody (sc-516102 Santa Cruz) dilution 1:4000 when used NF-kB p65 (G-8) antibody. The membranes were again washed three times for 5′ with PBS 1×, Tween 20 0.1% and once in PBS 1× alone. The signal was detected using ECL Prime (GE Healthcare) following the manufacturer's protocol.
Statistical analysis. All data (mean ± sem) were analyzed by a Student's t-test (two tailed) or by one-way ANOVA followed by Newman Keuls post hoc comparisons when appropriate.
Fluorescence microscopy. Cos-1 cells were transfected with GFP-p65 and GFP-p65 iso5 proteins on eight-well chamber slide (Biosigma) with DMEM (high glucose) supplemented with 10% heat inactivated fetal bovine serum. 24 h later cells were fixed with 4% paraformaldehyde in PBS for 10′ at room temperature and washed twice with PBS 1×. Nuclei were counterstained with 1 μg/ml of the nuclear stain Hoechst 33342 (Invitrogen) for 10′ at room temperature, the images were taken with digital Leica DMRA 2 microscope and 40× magnification.
Human liver and blood donor samples. Liver tissue and blood samples were collected from Caucasian patients. The liver samples were collected from a cohort of 14 patients (5 healthy subjects, 7 with cirrhosis and 2 with hepatocellular carcinoma), undergoing liver surgery at the Department of Laboratory Medicine and Advanced Biotechnologies, IRCCS-ISMETT (Istituto Mediterraneo per i Trapianti e Terapie ad alta specializzazione), Palermo, Italy. All tissue samples had been examined by a pathologist, and only healthy, cirrhotic, hepatocellular carcinoma histologically diagnosed liver tissues were used and liver proteins from every patient had been extracted. Blood samples from 42 patients (21 health, 21 SARS-CoV-2) were collected at the COVID Unit, Department of Internal Medicine, Hospital of National Relevance and High Specialization ARNAS Civico, Di Cristina, Benfratelli Palermo, Italy. Written informed consent was obtained from each patient studied.

Data availability
All data concerning the new exon sequences published on this study are deposited on data bank and will be available on NCBI gene bank after the possible acceptance of publication. p65 iso5 (GenBank accession number MN508965), p65 isoform 3 (p65 iso3) (GenBank accession number MN508964).