Cyclosporine A Inhibits Viral Infection and Release as Well as Cytokine Production in Lung Cells by Three SARS-CoV-2 Variants

ABSTRACT In December 2019, a new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) started spreading worldwide causing the coronavirus disease 2019 (COVID-19) pandemic. The hyperactivation of the immune system has been proposed to account for disease severity and death in COVID-19 patients. Despite several approaches having been tested, no therapeutic protocol has been approved. Given that Cyclosporine A (CsA) is well-known to exert a strong antiviral activity on several viral strains and an anti-inflammatory role in different organs with relevant benefits in diverse pathological contexts, we tested its effects on SARS-CoV-2 infection of lung cells. We found that treatment with CsA either before or after infection of CaLu3 cells by three SARS-CoV-2 variants: (i) reduces the expression of both viral RNA and proteins in infected cells; (ii) decreases the number of progeny virions released by infected cells; (iii) dampens the virus-triggered synthesis of cytokines (including IL-6, IL-8, IL1α and TNF-α) that are involved in cytokine storm in patients. Altogether, these data provide a rationale for CsA repositioning for the treatment of severe COVID-19 patients. IMPORTANCE SARS-CoV-2 is the most recently identified member of the betacoronavirus genus responsible for the COVID-19 pandemic. Repurposing of available drugs has been a “quick and dirty” approach to try to reduce mortality and severe symptoms in affected patients initially, and can still represent an undeniable and valuable approach to face COVID-19 as the continuous appearance and rapid diffusion of more “aggressive”/transmissible variants, capable of eluding antibody neutralization, challenges the effectiveness of some anti-SARS-CoV-2 vaccines. Here, we tested a known antiviral and anti-inflammatory drug, Cyclosporine A (CsA), and found that it dampens viral infection and cytokine release from lung cells upon exposure to three different SARS-CoV-2 variants. Knock down of the main intracellular target of CsA, Cyclophilin A, does not phenocopy the drug inhibition of viral infection. Altogether, these findings shed new light on the cellular mechanisms of SARS-CoV-2 infection and provide the rationale for CsA repositioning to treat severe COVID-19 patients.

Cyclosporine A (CsA), a natural cyclic peptide of 11 amino acids, is an inhibitor of cyclophilins (proteins belonging to the superfamily of immunophilins) known to prevent T cell activation via the formation of a tri-partite complex that includes Cyclophilin A (CyPA), CsA and calcineurin. The subsequent inhibition of NFAT translocation to the nucleus (14,15) and the inhibition of CyPA binding to interleukin-2 tyrosine kinase (Itk) that remains constitutively activated (16), reduces the immune response mediated by T cells (17). Moreover, CsA has been shown to affect also innate immunity (recently reviewed in [18]). Therefore, CsA exerts both immunosuppressive and anti-inflammatory activities.
We investigated the effects of CsA on SARS-CoV-2 infection in CaLu3 cells, a human pulmonary cell line; results showed that CsA hampers both viral infectivity and the production of pro-inflammatory cytokines by three different variants of SARS-CoV-2, suggesting a potential exploitation of this drug in the therapy of COVID-19.

RESULTS
SARS-CoV-2 is able to enter different organs, thus we tested four different human cell lines (A549 and CaLu3 from lungs, HepG2 from liver, and CaCo2 from intestine) as model systems to study viral infection, whereas we used Vero E6 cells (from African green monkey kidney, a standard system for laboratory propagation of viruses) to initially expand SARS-CoV-2. In accordance with previous findings (23), CaLu3 pulmonary cells were found to be the most efficiently infected and, therefore, used as the model system for all the experiments reported here.
Accordingly, immunofluorescence analysis confirmed that the number of infected CaLu3 cells was reduced by drug treatment in both experimental settings (Fig. S1), as revealed by anti-Spike protein labeling. Moreover, also the intracellular viral load, measured by ddPCR analysis of the RNA levels of nucleocapside (N1), was significantly decreased in cells treated with CsA either before (protocol 1, 1.32 6 0.19% of DMSO-treated samples) or after (protocol 2, 0.80 6 0.10% of DMSO-treated samples) infection with SARS-CoV-2 (Fig. 2B). To test whether CsA treatment also affects the production of an infectious progeny of SARS-CoV-2, we analyzed the levels of N1 RNA in the supernatant and quantified the virus titer by means of TCID 50 determination (Fig. 2C). We found that CsA treatment dampens the number of released infectious viral particles in both experimental conditions. Altogether, these findings show that CsA interferes with SARS-CoV-2 viral RNA replication, protein synthesis as well as with the assembly and release of new virions.
To test the efficacy of this drug at concentrations similar to those reached in the blood of transplanted patients (approximately 800 ng/mL [about 0.67 mM] in the first 2 to 4 h after administration-peak value-, and around 100 ng/ml [about 0.083 mM] 10-h postadministration-trough level_ [24]), we treated CaLu3 cells with 1 and 0.1 mM CsA (Fig. 3). ddPCR and TCID 50 analysis revealed that in both experimental settings all three concentrations of CsA reduce viral infection and release (with increasing efficacy augmenting drug dose), when compared with control DMSO-treated samples. In particular, CsA administration before infection appears more effective than after (protocol 1 vs protocol 2). Results also showed a good correlation between the levels of virus RNA in the cell extracts (Fig. 3A) and the number of released infectious viral particles (IVP) in the supernatants (Fig. 3C) of CsA-treated cells. On the other hand, we found higher levels of   Fig. 3C) in the supernatants in all the CsA-treated conditions, possibly due to non-infectious viral particle release and/or cell death.
Altogether, these results suggest that CsA is effective even at lower concentrations that are compatible with the clinical practice.
CsA exerts antiviral activity also on B.1.1.7 and P.1 SARS-CoV-2 variants. The appearance and rapid diffusion of more "aggressive"/transmissible variants of SARS-CoV-2, due to an increased affinity for ACE2 receptor and resistance to antibody neutralization (25,26), prompted us to test the efficacy of CsA also on these new strains. In particular, we analyzed the viral load in samples treated with the three concentrations of CsA after infection (protocol 2) with either B.1.1.7 (U.K variant, alpha) or P.1 (Brazil lineage, gamma) SARS-CoV-2 by ddPCR and found that CsA treatment significantly reduced virus RNA replication in the cells at all the concentrations tested for both SARS-CoV-2 strains (Fig. 4A). Results showed a good correlation between the levels of viral RNA and IVP in the supernatants of CsA-treated B.1.1.7-infected cells, suggesting that this variant releases mainly infecting virions. By contrast, we found higher levels of viral RNA (Fig. 4B) compared with IVP ( Fig. 4C) in the supernatants of CsA-treated P.1-infected cells, possibly due to non-infectious viral particle release and/or cell death.
Altogether, these results show that CsA exerts antiviral activity also on SARS-CoV-2 P.1 and B.1.1.7 variants. Moreover, our findings suggest that the lowest (0.1 mM) CsA concentration is more effective on the EU strain than on the SARS-CoV-2 variants ( Fig. 3 and 4).
CsA diminishes the synthesis of virus-induced cytokines in lung cells. SARS-CoV infection was reported to induce the synthesis of epithelial cytokines in CaLu3 cells (27); therefore we decided to investigate whether also SARS-CoV-2 triggered cytokine production and to test the effect of CsA treatment. To this purpose we analyzed the levels of a panel of relevant cytokines, including IL1a (which triggers the recruitment of hematopoietic cells that in turn amplify and induce IL1a production in a positive feedback loop sustaining inflammation [28]), TNF-a (a strong pro-inflammatory cytokine [29]), IL-8 (which plays an important role in both neutrophil recruitment and activation [30]), and IL-6 (which exerts pro-inflammatory activities in a context-dependent manner [31]), which are involved in the cytokine release disease reported in COVID-19 patients, in the cellular extracts of CaLu3 treated with CsA either before (protocol 1) or after (protocol 2) SARS-CoV-2 infection by rtPCR analysis. Results showed that CsA significantly reduced the amount of cytokine RNA synthesized upon viral infection in both experimental settings and by all SARS-CoV-2 strains at the higher concentrations tested (10 and 1 mM, Fig. 5). Moreover, we found that CsA appears to exert an "all or none" effect on cytokine RNA production induced by B.1.1.7 and P.1 variants (effective at similar levels at 10 and 1 mM and almost ineffective at 0.1 mM), whereas the drug displays a more dose-dependent effect on cytokine induction promoted by the EU strain of SARS-CoV-2.
Altogether, these findings support an anti-inflammatory activity of CsA on lung cells infected by SARS-CoV-2. Cyclophilin A silencing does not phenocopy CsA effects on SARS-CoV-2 infection. CyPA is the main intracellular target of CsA (32) and it acts as an intracellular: (i) chaperone during viral replication for different viruses (33,34); and (ii) sensor that favors viral infection (35,36). Furthermore, CyPA has been identified as the top ranked hit in a meta-analysis study of host genes implicated in COVID-19 (37), and CsA ability to impair SARS-CoV-2 infection in CaLu3 cells has been suggested to depend on its action on cyclophilins (23). Thus, we investigated the role of CyPA in SARS-CoV-2 entry into host cells, exploiting a genetic approach. We silenced CyPA expression in CaLu3 cells before viral infection by transduction with a specific short hairpin RNA (shRNA): CyPA knock down efficiency was 96.0% 6 0.7% of control NT shRNA-transduced cells at the RNA and 95.1% 6 1.0% of control NT shRNA-transduced cells at the protein level. We evaluated SARS-CoV-2 RNA load in both cells and supernatants by ddPCR analysis, and found that N1 levels were increased in CyPA-knocked down cells (166.3% 6 6.1% and 156.1% 6 11.6% of control NT shRNA-transduced cells, respectively, Fig. 6A). Furthermore, we assessed the levels of viral Spike protein by WB analysis (Fig. 6B and C), and found that silencing of CyPA augmented its levels (280.6% 6 10.7% of control NT shRNA-transduced cells, Fig. 6C). To test whether knock down of CyPA also affects the production of an infectious progeny of SARS-CoV-2, we quantified the virus titer by means of TCID 50 determination and found that CyPA silencing increases the number of released IVP (Fig. 6D).
Because reduction of viral infection impairs cytokine production (Fig. 5), we analyzed the RNA levels of IL1a, IL-6, IL-8, and TNF-a in Calu3 cells treated with CyPA-specific shRNA before viral infection, and found that CyPA knockdown augmented their expression (IL1a, 757.1 6 129.9; TNF-a, 843. Altogether, these results suggest that CyPA works as a negative modulator of SARS-CoV-2 infection, and therefore, that CsA inhibitory effects on both viral replication and cytokine production have to be ascribed to a different molecular mechanism.

DISCUSSION
In December 2019, the newly identified coronavirus SARS-CoV-2 started spreading worldwide causing the COVID-19 pandemic. Several approaches have been suggested and tested for the treatment of COVID-19 patients, but till now no therapeutic protocol has been approved. The lack of solid therapeutic approaches for the treatment of COVID-19 patients led to the idea of verifying whether CsA, a molecule endowed with potent antiviral and anti-inflammatory activities, could play a role in this scenario (38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48)(49).
Some preliminary data obtained in Vero E6 cells suggested that CsA might interfere with SARS-CoV-2 infection (50); thus we tested the drug effects in the physiological context of pulmonary CaLu3 cells. We found that in vitro CsA exerts two different effects: (i) it impairs viral infection, replication and release, and (ii) it diminishes the virusinduced synthesis of cytokines by CaLu3 cells (as previously reported for SARS-CoV [27]). Recently, the ability of CsA to interfere with SARS-CoV-2 infection has been reported also in CaLu3 cells, and suggested to rely on its action on cyclophilins (23). Here, we provide additional evidence that CsA interferes with viral infection and dampens subsequent epithelial cytokines production also by B.1.1.7 (alpha) and P.1 (gamma) variants of SARS-CoV-2, thus suggesting that the "emerged" viral mutations have not affected the drug-targeted molecular machinery. Moreover, by exploiting a genetic approach, we show that CsA effect is not mediated by its main intracellular target, CyPA.
CsA is known to exert several antiviral activities, including the inhibition of genome replication and particles assembly, as well as the regulation of the activity of host restriction factors (20,21), mainly by its inhibitory functions on cyclophilins, in particular CyPA. Indeed, it plays an essential role in promoting viral infection exerting its functions both inside and outside host cells. In particular, CyPA (i) acts as an intracellular chaperone during viral replication for different viruses (33,34); (ii) behaves as an intracellular sensor that favors viral infection (hampering the innate immune response [35], and regulating the sensitivity to host restriction factors [36]); and (iii) partakes in target cells invasion by HIV-1 and SARS-CoV (51-53), but not by SARS-CoV-2 (54), mediating virus binding to the CD147 receptor. Furthermore, CyPA has been recently reported as the top ranked hit in a meta-analysis study of host genes implicated in COVID-19 (37). Interestingly, our experiments with CyPA-silenced cells provide evidence that CyPA works as a negative modulator of SARS-CoV-2 infection, as already described for influenza virus and rotavirus (33,34). Therefore, our results suggest that CsA ability to impair viral activity has to be ascribed to other cyclophilins or to a different molecular mechanism.
As for the former hypothesis, two additional candidates should be considered among cyclophilins, namely, CyPB and CyPD. CyPB (found in the endoplasmic reticulum) is the only other cyclophilin expressed at detectable levels inside the cells in addition to CyPA, which instead accounts for 0.1% to 0.5% of the total cellular protein content. CyPB has been reported to play a role in host infection by HCV (55) and HIV-1 (56), in the latter case modulating viral translocation into the nucleus in a CsA-independent manner (57). CyPD, an immunophilin localized on the inner membrane of mitochondria, has been shown to partake in coronavirus (HCoV-OC43) infection in a CsA-dependent manner (58,59).
As for the latter hypothesis, CsA has been proposed recently to be able to interfere with SARS-CoV-2 entry. Indeed, Prasad et al. unraveled that CsA can bind and inhibit two classes of host proteases, namely, TMPRSS2 and Cathepsins, using a computational approach, and suggested that the drug might work also on the initial phases of SARS-CoV-2 infection of target cells (60). Moreover, some data provided by Dittmar et al. could support an involvement of TMPRSS2 in CsA mechanism of action as they show that cell treatment with camostat appears to phenocopy the effects of CsA on SARS-CoV-2 infection (23). Our findings that CsA functions in both experimental settings (protocol 1, CsA treatment before infection, and protocol 2, CsA treatment after infection) are compatible with either hypothesis. Indeed, CsA is a membrane permeable drug, thus it might work on already infected cells inhibiting new virions assembly and/or blocking new cell infection by viral progeny.
CsA exerts anti-inflammatory activities as well, both inside and outside the cell. Intracellularly, CsA sequesters cyclophilins from binding to calcineurin, thus avoiding NFAT translocation to the nucleus and downstream cytokine synthesis (18). Extracellularly, CsA binds secreted CyPA or CyPB and impairs their chemotactic activity on eosinophils, neutrophils, T lymphocytes which is driven by recognition of the CD147 receptor (61,62), and dampens the inflammatory response in an animal model of human acute lung injury (63). Altogether, these observations suggest that extracellular CyPA plays an essential role in inflammation in different contexts, and that its targeting might represent an effective way to reduce leukocyte redistribution to inflamed tissues and local production of cytokines (64). This hypothesis is strengthened by the finding that under mechanical ventilation airway epithelial cells actively secrete CyPA that is responsible for cytokine-driven leukocytemediated acute lung injury in mice, and that this phenotype is reverted upon treatment with a cyclosporine derivative (65). Moreover, extracellular CyPA levels have been found upregulated in the bronchoalveolar lavage fluids of patients with ARDS (65).
Thus, the anti-inflammatory activity of CsA might be useful to dampen the cytokine storm, possibly delaying the progression toward acute respiratory distress syndrome (ARDS) and/or a systemic inflammatory condition as observed in severe COVID-19. Indeed, patients display distinct hematological manifestations; among them lymphocytopenia characterizes approximately 70% of severe-to-critical cases. Different causes have been proposed to account for this clinical manifestation: immune exhaustion/senescence, massive recruitment of immune cells to tissues, and decreased cell production due to uncoordinated cytokine signaling (66)(67)(68). The common denominator of all these phenomena relies on the hyperactivation of the immune system and an uncoordinated excessive cytokine signaling. In this context, our finding that CsA attenuates cytokine synthesis in lung cells could result in a reduced recruitment of immune cells to the site of infection. Indeed, regulators of the immune function have been employed as therapeutic approaches (i.e., corticosteroids, anti-IL-17 monoclonal antibodies, anti-IL-6 monoclonal antibodies). Although they are known to induce lymphocytopenia, corticosteroids can promptly recover lymphocytes count due to their anti-inflammatory properties (69). In addition, they reduce mortality and the need for invasive mechanical ventilation or oxygen alone (possibly because they promote erythroid precursors maturation into red blood cells [70]), but only in severe COVID-19 patients (71,72), whereas treatment with dexamethasone does not show beneficial effects in patients not requiring respiratory support (72) or even an increased risk of mortality or mechanical ventilation need in patients with low levels of initial C-reactive protein (71).
As for cytokine-targeted therapies, results of randomized, placebo-controlled, blinded trials targeting either IL-1 or IL-6 pathway showed no survival benefit in COVID-19 patients ( [73] and [74], respectively), unless performed in combination with cortisteroids (75,76), suggesting that inhibiting a single cytokine pathway might not be sufficient. In this context, our findings showing that CsA dampens the production of several epithelial cytokines would support its repurposing. Furthermore, our observation of a reduction of viral titer as well as of cytokine production at concentrations compatible with CsA administration to patients would support its usage in vivo (as recently questioned by Solanich et al. [77]).
Despite we are well aware of its potent immunosuppressive activity, we reckon that repositioning of CsA should be considered for the treatment of COVID-19 patients, upon the identification of the proper/best therapeutic window (78,79). Indeed, some clinical evidences have been collected retrospectively on the treatment of severe COVID-19 patients with CsA; results showed no additional risks in face of reduced mortality (80). Along this line, a phase I clinical trial testing the ability of CsA to prevent cytokine storm onset in patients with moderate COVID-19 has been started in 2020 and is still ongoing (NCT04412785).
Our findings showing that CsA exerts both antiviral and anti-inflammatory activities on three different variants of SARS-CoV-2 with similar efficacy would suggest that this drug exploits a conserved mechanism and therefore might be useful in the therapy of COVID-19. By contrast, the effectiveness of some anti-SARS-CoV-2 vaccines appears to be reduced by certain viral mutations and thus novel alternative therapeutic approaches might be essential in the clinical management of COVID-19 patients (81)(82)(83)(84)(85). Finally, the well-known inhibitory activity of CsA on different viruses together with the results reported here demonstrating its efficacy on different SARS-CoV-2 variants support the relevance of this drug beyond the current pandemic.

MATERIALS AND METHODS
Antibodies and reagents. Antibody anti-CD147 was from Santa Cruz Biotechnology (Dallas, TX, USA). Antibody anti-Spike protein antibody was from Genetex (Alton Pkwy Irvine, CA, USA). Antibodies anti-ACE2, anti-GAPDH, and anti-CyPA were from Abcam (Cambridge, Cambridgeshire, UK). HRP-conjugated secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) unless otherwise stated. Hoechst 33542, Alexa 488-, and Alexa 546-conjugated secondary antibodies were obtained from Molecular Probes (Life Technologies by Thermo Scientific, Waltham, MA, USA). CsA, DMSO, HEPES, MTT, Tris, Glycine, SDS, Tween 20, saponin, NH 4 Cl, and bovine serum albumin (BSA) were purchased from Sigma (by Merck, Kenilworth, NJ, USA). All cell culture reagents were from Thermo Scientific. All chemical reagents were of analytical grade or higher, and purchased from Sigma unless otherwise specified.
SARS  Table 1 for features of the viral strains used in this study). All the experiments with SARS-CoV-2 virus were performed in BSL3 facility (Department of Biomedical and Clinical Sciences "L. Sacco," Milano University Medical School); virus was inactivated according to institutional safety guidelines, before samples analysis outside BSL3 area. In order to obtain the viral stock, SARS-CoV-2 was expanded on the human cell line CaCo2 and infectious viral particles concentration was assessed by TCID 50 . Briefly, Calu3 were seeded at 2 Â 10 4 cells per well in a 96well plate. Eleven 1:10 serial dilutions of the viral stock were performed in 2% FBS medium. For each dilution, eight wells were infected. Eight wells were left uninfected as control. Three-hours postinfection (hpi), each well was thoroughly washed three times with pre-warmed PBS and the culture media replaced with 10% FBS DMEM. Optical microscope observation (ZOE Fluorescent Cell Imager, Bio-Rad Laboratories, Hercules, CA, USA) was performed daily to investigate the cytopathic effect. At 48-hpi, supernatants were removed, cells fixed by paraformaldehyde (PFA) 4% for 1 h at room temperature, then stained by 0.2% crystal violet solution. By applying the Reed-Muench method with the correction for the proportional distance (PD [86]), we were able to assess the TCID 50 and to calculate the MOI in our experiments.
The day before, 2.5 Â 10 5 Calu3 cells were cultured in 0.5 mL of 2% FBS medium in a 24-well plate. Then, we followed two different protocols of infection, as follows: Protocol 1: Cells were first pretreated with 10, 1, or 0.1 mM CsA (or equivalent DMSO, as mock control) diluted in complete medium in the absence of the virus. After 4 h of pretreatment, cells were challenged with 0.05 MOI of SARS-CoV-2. At 3-hpi, cells were thoroughly washed three times with pre-warmed PBS and refilled with the complete growth medium (10% FBS), including 10 mM CsA, DMSO, or plain culture medium (Fig. 1A).
At 48-hpi, CaLu3 cells were lysed for RNA or protein extraction, whereas supernatants were harvested and appropriately stored.
Proliferation assay. Cells were seeded in a 96-well plate and allowed to grow for 48 h before treatment. Cell viability was determined by the MTT assay after 48 h of treatment. Cells were incubated with 2 mM 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma) for 4 h at 37°C; then the supernatant was removed. Afterwards, formazan was extracted from cells with 100 mL of DMSO. The amount of MTT-formazan was determined by absorbance at 595 nm.
RNA interference. To knockdown CyPA expression, a short hairpin sequence targeting human CyPA and a non-targeting (NT) negative control shRNA were used, as described before (87).
The CyPA-specific oligonucleotide used was: 59-CTGACTGTGGACAACTCGAAT-39. The NT shRNA used was: 59-CAACAAGATGAAGAGCACCAA-39. Briefly, for lentivirus production, Hek293T cells were transfected with the calcium phosphate method. To this end, a mix containing 10 mg of either lentiviral plasmid DNA vector, 6.5 mg of packaging vector Dr 8.74, 3.5 mg of Env VSV-G, 2.5 mg of REV, ddH 2 O to 450 mL, 50 mL of 2.5 M CaCl2, and 500 mL of 2x HBS was added dropwise over a monolayer of Hek293T cells seeded on a 10-cm 2 dish. After 16 h, the medium was replaced. Thirty hours later, the medium containing virus particles was collected and passed on a 0.22-mm filter. CaLu3 cells were infected overnight and the following day selected with 1.5 mg/mL puromycin (Thermo Scientific) treatment for 10 days. Transduced cells were infected with SARS-CoV-2 on day 12 and samples collected for analysis on day 14.
RNA extraction and reverse transcription. Cell supernatant was collected and Maxwell RSC Viral Total Nucleic Acid purification kit was used to extract RNA from 250 mL of cell culture supernatants employing the Maxwell RSC Instrument (Promega, Madison, WI, USA). The remaining supernatant was conveniently stored at 280°C for the TCID 50 assessment. Each well was then thoroughly washed three times with pre-warmed PBS. Cells were lysed and collected in 100 mL of RNAzol (TEL-TEST, Inc., Friendswood, TX, USA). RNA extraction was performed employing the acid guanidium-phenol-chloroform (AGPC) extraction method, as elsewhere described (88). One mg of total RNA was reversed transcribed in a final volume of 20 mL using the Reverse Transcription Kit (Promega). Target cDNA was amplified by either ddPCR or rtPCR.
Results were expressed as relative expression units (nFold) to the housekeeping reference gene (GAPDH) calculated by the 2 2DDCt method.
Western blotting. For WB analysis, cells were lysed directly in 2x Laemmli buffer in order to inactivate the virus and to be able to process them outside BSL3 area. Samples were boiled for 5 min at 95°C before loading onto the gel. Proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes (Hybond, GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK). Strips containing the proteins of interest were incubated in 5% (wt/vol) BSA in TBS containing 0.1% (vol/vol) Tween 20, pH 7.4 (T-TBS), for 1 h at room temperature and then with fresh blocking buffer containing the primary antibody at its working concentration (see Table 3). After overnight incubation at 4°C, the antibodies were removed and the strips washed with T-TBS for 3 Â 10 min. Strips were incubated for 1 h with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody and washed 3 Â 10 min with T-TBS. WBs were developed using the chemiluminescent method (ECL, GE Healthcare) and signals acquired by ChemiDoc MP Imaging System (Bio-Rad Laboratories). Bands were quantified by densitometric analysis using the National Institutes of Health (NIH) ImageJ program. The quantification of each band was normalized using the signal of housekeeping proteins (CyPA or GAPDH) as a loading control.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only. SUPPLEMENTAL FILE 1, PDF file, 0.4 MB.

ACKNOWLEDGMENTS
We thank Davide Mileto from ASST Fatebenefratelli-Sacco for kindly providing the B.