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Definitions

• the present invention concerns anti-BAG3 antibodies for use as biochemical markers in the diagnosis of a pathological state.
• BAG3 (RefSeq: NP_ 004272; Gene ID 9531) is a 74 kDa cytoplasmic protein particularly concentrated in the rough endoplasmic reticulum.
• BAG3 protein belongs to the family of co-chaperones that interact with the ATPase domain of the heat shock protein HSP70 through the structural domain known as BAG domain (110-124 amino acids) .
• BAG3 contains a WW domain and a proline-rich repeat (PXXP) , that can mediate binding to other proteins.
• PXXP proline-rich repeat
• bag3 gene expression is constitutive in a few normal cell types, including myocytes, and in several primary tumours or tumour cell lines. Moreover it can be induced by a variety of stressors: indeed stressful stimuli activate the heat shock transcription factor (HSF) 1, that is responsible for the expression of stress- activated genes, including bag3 (Rosati A, Graziano V, De Laurenzi V, Pascale M, Turco MC .
• HSF heat shock transcription factor
• BAG3 a multifaceted protein that regulates major cell pathways. Cell Death Dis. 2011; 2: el41) .
• Evidence indicates that BAG3 has a role in sustaining cell survival, by modulating, in either Hsp70- dependent or -independent fashion, the levels or localisation of apoptosis- regulating proteins, such as ⁇ , Bax or BRAF, depending on cell context .
• BAG3 protein appears to be expressed during cardiomyoblasts differentiation and to sustain myogenin expression. These findings indicate an involvement of BAG3 in late heart development (De Marco M, Turco MC, Rosati A. BAG3 protein is induced during cardiomyoblast differentiation and modulates myogenin expression. Cell Cycle. 2011; 10: 850-852). Moreover, in cardiomyocytes BAG3 has been shown to localize at Z-disc and interact with the actin capping protein, CapZpl, stabilizing myofibril structure and possibly preserving myofibrillar integrity during mechanical stress. BAG3 mutations can impair the Z-disc assembly and increase the sensitivity to stress-induced apoptosis.
• the present invention concerns anti-BAG3 antibodies for use as biochemical markers in the diagnosis of a pathological state.
• anti-BAG3 antibodies are bound to soluble BAG3 to form immune complexes.
• said diagnosis is in vitro or ex vivo .
• a further aspect of the present invention is that the recipient of said diagnosis is a mammalian, preferably a human.
• the use of the anti-BAG-3 antibodies of the present invention has the advantages of being specific for a pathological state selected from the group consisting of a heart disease, cancer, diabetes, inflammation and inflammatory related diseases of the skin, nerves, bones, blood vessels and connective tissues.
• said heart disease is selected from: angina pectoris, pre-infarction angina, myocardial infarction, heart failure, ischemia, acute coronary disease, acute heart failure, chronic heart failure and iatrogenic heart disease.
• said cancer is selected from: pancreatic cancer, bladder cancer and prostate cancer.
• a further embodiment of the present invention is a method for detecting the presence of an anti- BAG3 antibody or an anti-BAG3 antibody bound to soluble BAG3 to form an immune complex in a biological sample, comprising the steps of:
• the method of the present invention further comprises the additional step of :
• said determination step b. is performed by an ELISA test.
• said serum, plasma, urine or saliva is from a mammalian, preferably a human.
• the presence of said anti-BAG3 antibody or said immune complex is associated with a pathological condition.
• said pathological condition is selected from the group consisting of a heart disease, cancer, diabetes, inflammation and inflammatory related diseases of the skin, nerves, bones, blood vessels and connective tissues .
• said heart disease is selected from: angina pectoris, pre-infarction angina, myocardial infarction, heart failure, ischemia, acute coronary disease, acute heart failure, chronic heart failure and iatrogenic heart disease .
• said cancer is selected from: pancreatic cancer, bladder cancer and prostate cancer .
• a further embodiment of the present invention is an ELISA kit, comprising a BAG3 recombinant protein or BAG3-specific mouse monoclonal antibodies AC-1, AC-2 AC-3, AC-4, AC-5, AC-rbla, AC-rblb, AC-rb2a, AC-rb2b, AC-rb3a, AC-rb3b, AC- rb4a and AC-rb4b for capturing soluble BAG3 and antibodies able to recognize human immunoglobulins, as well as its use for the detection of anti-BAG3 antibodies or anti-BAG3 antibodies bound to soluble BAG3 to form an immune complexes in a biological sample.
• a BAG3 recombinant protein or BAG3-specific mouse monoclonal antibodies AC-1, AC-2 AC-3, AC-4, AC-5, AC-rbla, AC-rblb, AC-rb2a, AC-rb2b, AC-rb3a, AC-rb3b, AC- rb
• said biological sample is a serum, plasma, urine or saliva sample.
• said serum, plasma, urine or saliva sample is from a mammalian, preferably a human.
• the invention still further relates to a immunohistochemistry (IHC) kit for the detection of BAG3 protein in a biological sample, wherein said biological sample is preferably a tissue sample, comprising BAG3- specific antibodies and reagents including probes needed for the staining .
• IHC immunohistochemistry
• FIG. 1A detection of BAG3 protein in supernatants from cultured cardiomyocytes .
• Human (HCMa) and rat (H9c2) cardiomyocytes at 80% confluency were incubated with or without 10% FBS for 16 hours at 37°C in a 5% CO 2 atmosphere.
• Figure IB detection of BAG3 protein in exocytic vesicles.
• Surnatants obtained from H9c2 cells were subjected to sequential centrifugations : (i) 2' 000xg for 15 min, to remove cells; (ii) 10'OOOxg for 30 min, to remove cellular debris; (iii) 150' 000xg for 90 min, to pellet exocytic vesicles. The pellet was washed once in PBS at 150' 000xg for 90 min and analyzed with the anti- BAG3 TOS-2 polyclonal antibody in comparison with a whole-cell lysate by western blot. Rab-4 was analyzed as a marker for exocytic vesicles.
• GAPDH a cytosolic protein, was analyzed as a control .
• Figure 1C Sera from two healthy donors and from two patients affected by chronic heart failure were analyzed with the anti-BAG3 antibody TOS-2 polyclonal antibody in western blotting.
• FIG. 2A detection of BAG3 protein in sera from CHF patients.
• BAG3 recombinant protein and whole- cell lysate from HCMa cells were analyzed by western blotting with serum (1:40) obtained from a patient with heart failure. Analysis with serum from a healthy donor was performed as negative control .
• Figure 2B detection of anti-BAG3 antibodies by ELISA test. Sera from 50 CHF patients (with ejection fraction ⁇ 60%) were compared with sera from 50 healthy donor for the presence of anti- BAG3 antibodies in a specific ELISA test. Results are plotted as arbitrary units.
• Figure 2C ROC analysis of ELISA test results. Cut-off on 0.083 A.U. results in 74% sensitivity and 68% specificity.
• FIG. 3A Confocal microscopy analysis of direct fluorescence performed for detection of rBAG3- FITC binding to HCMa cells (a, b, c) and J774 Al cells (d, e, f, g, h, i) .
• BAG3 recombinant protein and purified BSA albumin from bovine serum purchased from SIGMA
• BSA-FITC Conjugation Kit purchased from SIGMA following the manufacturer instructions.
• ⁇ -integrin was analyzed as control (a, d, g) .
• Cells were analyzed by a Zeiss LSM confocal microscope. Merged images are shown in c, f and i .
• FIG. 3B BAG3 binds macrophages. J774 Al macrophages (lxlO 6 cells/ml) were incubated with different concentration of Fitc-BAG3 protein (7, 14 and 70 nm) . FITC-BSA (70 nM) was used as a negative control (grey) . Cells fluorescence was analyzed by flow cytometry.
• J774 Al cells at 80% confluency were incubated with control medium, BSA, LPS or rBAG3, for 20 hours.
• Polymixin was added where indicated to verify that the effects of E. coli- derived rBAG3 were independent from the presence of contaminating endotoxin.
• Cox-2 and iNOS expression were analyzed in cell lysates by Western blotting.
• J774 Al cells at 80% confluency were incubated with control medium, BSA, LPS or rBAG3 for 24 hours.
• 100 ⁇ of supernatants from each sample were incubated with 100 ⁇ of Griess reagent; the optical density at 550 nm (OD550) was measured with a Beckman DU62 spectrophotometer.
• Nitrite concentration was evaluated by comparing the OD550 of the sample with that of a standard curve of sodium nitrite.
• FIG. 3C panel c - Analysis of IL-6 release from J774 Al macrophages incubated with BAG3.
• J774 Al cells at 80% confluency macrophages were incubated with control medium, BSA, LPS or recombinant BAG3 for 5 hours.
• BAG3 peptides peptide 1, peptide 2, peptide 3, peptide 4 or scrambled peptide 625 nM were added where indicated to verify their ability to block BAG3 activity.
• IL-6 production was measured in cell culture medium using an ELISA test.
• IL-6 concentration was evaluated by comparing the OD of the sample with that of a standard curve of recombinant IL-6.
• FIG. 5 Representative image of BAG3 staining in synovial tissues from several rheumatoid arthritis. BAG3 positivity is observed in synovial fibroblasts and inflammatory infiltrates. Sections were counterstained with hematoxylin.
• bag3 mRNA relative expression evaluated by qRT- PCR is shown in a graph where values are reported as mean + S.D.
• the blue line represents the median value calculated.
• Pancreatic cancer cell lines (PSN1, Capan-1, AsPC-1, PANC-1 and MIA PaCa-2 ) were treated with different concentrations of gemcitabine as indicated in the graph. After 48 hours, apoptotic cell death was analyzed. Graph depicts mean percentage of Sub G0/G1 cells ( ⁇ S.D.). Data are representative of three independent experiments.
• Figure 8B
• MIA PaCa-2 and PANC-1 cell lines were treated with 2 ⁇ gemcitabine (GEM) for the indicated times BAG3 protein expression levels were monitored by western blot .
• GEM gemcitabine
• bag3 mRNA levels were analyzed by RT-PCR; graph depicts relative bag3 mRNA levels ( ⁇ S.D.) and data are representative of three indipendent experiments .
• MIA PaCa-2 and PANC-1 cell lines were transfected with BAG3 siRNA or a non- targeted siRNA (NTsiRNA) for 72 hours and then treated with 2 ⁇ gemcitabine (GEM) for 24h.
• BAG3 levels were analyzed by western blot and GAPDH levels were detected to monitor equal loading conditions.
• MIA PaCa-2 and PANC-1 cells were transfected as described above and treated with 2 ⁇ gemcitabine (GEM) for 24h or 48h. Apoptotic cell death was analyzed as described. Graph depicts mean percentage of Sub G0/G1 cells ( ⁇ S.D.). Data are representative of three independent experiments.
• Figure 9A Detection of BAG3 specific immuno- complexes by ELISA test. Sera from 55 pancreatic adenocarcinoma patients were compared with sera from 51 healthy donors for the presence of BAG3 specific immune-complexes in a specific ELISA test. Results are plotted as arbitrary units +S . E .
• Figure 9B ROC analysis of ELISA test results. Cut-off on 0.183 A.U. results in 65% sensitivity and 78% specificity.
• Detection of BAG3-specific antibodies in patients sera from 49 female subjects at 0-1 years pre- diagnosis for pancreatic adenocarcinoma were compared with sera from 235 female control subjects sera for the presence of BAG3 specific antibodies in a specific ELISA test. Control sera were about five for each case subjects and age matched with the respective case. Others time points included: 53 subjects at 1-2 years pre- diagnosis for PDAC compared to 251 controls; 44 subjects at 2-3 years pre-diagnosis for PDAC compared to 212 controls; 42 subjects at 3-4 years pre-diagnosis for PDAC compared to 187 controls .
• Results are plotted as arbitrary units _+S . P value was calculated with student's t test.
• the present invention concerns anti-BAG3 antibodies for use as biochemical markers in the diagnosis of a pathological state.
• diagnosis in the present invention we refer to medical diagnosis (often simply termed diagnosis) that refers to the process of attempting to determine or identify a possible disease or disorder.
• diagnosis of the present invention encompasses also the early diagnosis
• Anti-BAG3 antibodies have now been advantageously detected in serum, plasma, urine or saliva. Until now such antibodies had never been found in serum either in physiological or pathological condition.
• the detection of anti-BAG3 antibodies in serum has the advantage of being a rapid and non-invasive technique be exploited for diagnostic, early diagnosis and prognostic purposes, risk stratification, as a tool for the identification and for monitoring therapies.
• a further advantage of the detection of antibodies is that a very small amount of serum is required for the detection.
• soluble BAG3 protein can also be detected in the serum of patients suffering from some pathologies, but the amount of serum requested for the detection of the soluble protein is much higher than that required for the detection of antibodies.
• soluble BAG3 protein levels can be much lower than those of antibodies and/or that, respect to soluble BAG3 protein, antibodies can be detectable in earlier phases of specific pathologies and/or can more efficiently predict risk of complications or monitor the effects of therapies.
• a further embodiment of the present invention is that said anti-BAG3 antibodies can be advantageously detected in different biological samples selected from serum, plasma, urine or saliva .
• serum is intended the component of blood that is neither a blood cell nor a clotting factor; it is the blood plasma with the fibrinogens removed.
• Serum includes all proteins not used in blood clotting (coagulation) and all the electrolytes, antibodies, antigens, hormones, and any exogenous substances .
• plasma is intended the straw-coloured/pale-yellow liquid component of blood that normally holds the blood cells in whole blood in suspension. It contains clotting factors, such as fibrinogens.
• the invention provides the use of anti-BAG3 antibodies as biochemical markers, wherein said anti-BAG3 antibodies are bound to soluble BAG3 to form immune complexes.
• Anti-BAG3 antibodies either free or bound to soluble BAG3 to form immune complexes have advantageously been now detected in a biological sample, and may be used as a marker in the diagnosis of a pathological condition.
• the detection of such antibodies and/or immune complexes in a biological sample also has the advantage of being a rapid and non-invasive technique for diagnostic and/or prognostic purposes .
• said diagnosis is in vivo or ex vivo .
• a further embodiment of the present invention is that recipient of said diagnosis is a mammalian, preferably a human.
• immune complex or protein/antibody complex is intended the integral binding of an antibody to a soluble antigen, the bound antigen acting as a specific epitope, bound to an antibody is referred to as a singular immune complex.
• Immune complexes have the same advantages seen as for the detection of antibodies, since a very small amount of serum is required also for the detection of the immune complex.
• the amount of serum required for the detection of soluble BAG3 is much higher also than that required for the detection of the protein/antibody (immune) complexes.
• Immune complexes as well as free antibodies can be detectable in earlier phases of specific pathologies and can more efficiently predict risk of complications or monitor the effects of therapies.
• a still further embodiment of the invention is the use of anti-BAG3 antibodies or said immune complexes (formed by anti-BAG3 antibodies bound to soluble BAG3 ) as biological markers of a pathological state, wherein said pathological state is a heart disease, cancer, diabetes, inflammation and inflammatory related diseases of the skin, nerves, bones, blood vessels and connective tissues.
• said heart disease is selected from the group consisting of: angina pectoris, preinfarction angina, ischemia, myocardial infarction, heart failure, acute coronary disease, acute heart failure, chronic heart failure and iatrogenic heart disease.
• said cancer is selected from: pancreatic cancer, bladder cancer and prostate cancer .
• a further embodiment of the present invention is a method for detecting the presence of an anti- BAG3 antibody or an anti-BAG3 antibody bound to soluble BAG3 to form an immune complex in a biological sample, comprising the steps of:
• the method according to the present invention has the advantage of allowing to detect significant differences between anti-BAG3 antibodies and/or BAG3 /antibodies complexes between healthy individuals and patients affected by BAG3- involving pathologies.
• the proposed assay method allows a statistically significant separation of the group of cardiac patients from the group of healthy people. It can also stratify such patients with heart disease in subgroups of patients at increased risk (heart failure, HF).
• HF heart failure
• the method according to the present invention is a method wherein said determination step b. is performed by an ELISA test .
• said serum, plasma, urine or saliva is from a mammalian, preferably a human .
• the method according to the present invention has the advantage of allowing the rapid and noninvasive detection of the biological markers allowing the evaluation of pathologies, risks for diseases and/or their complications, and monitoring of therapies.
• the invention relates to a detection method wherein the presence of said anti-BAG3 antibody or said anti- BAG3 antibody bound to soluble BAG3 to form an immune complex is associated with a pathological condition .
• said pathological condition is chosen from the group consisting of heart disease, cancer, diabetes, inflammation and inflammatory related diseases of the skin, nerves, bones, blood vessels and connective tissues.
• said heart disease is selected from the group consisting of: angina pectoris, pre-infarction angina, myocardial infarction, ischemia, heart failure, acute coronary disease, acute heart failure, chronic heart failure or iatrogenic heart disease.
• said cancer is selected from: pancreatic cancer, bladder cancer and prostate cancer .
• the invention further relates to an ELISA kit for the detection of anti-BAG3 antibodies or anti- BAG3 antibodies bound to soluble BAG3 to form an immune complexes in a biological sample.
• the ELISA kit according to the present invention comprises a BAG3 recombinant protein for capturing anti-BAG3 antibodies or BAG3- specific mouse monoclonal antibodies AC-1, AC-2, AC-3, AC- 4, AC-5, AC-rbla, AC-rblb, AC-rb2a, AC-rb2b, AC- rb3a, AC-rb3b, AC-rb4a and AC-rb4b, for capturing soluble BAG3 and antibodies able to recognize human immunoglobulins.
• Such antibodies can be enzyme-linked antibodies able to recognize human immunoglobulins.
• the invention also relates to a kit for the detection of BAG3-associated antibodies in a biological sample and is performed by ELISA with BAG3-specific mouse monoclonal antibodies AC-1, AC-2, AC-3, AC-4, AC-5, AC-rbla, AC-rblb, AC- rb2a, AC-rb2b, AC-rb3a, AC-rb3b, AC-rb4 and AC- rb4b, for capturing soluble BAG3 and enzyme- linked antibodies for the detection able to recognize human immunoglobulins.
• said biological sample is a serum, plasma, urine or saliva sample.
• said serum, plasma, urine or saliva sample is from a mammalian, preferably a human.
• the invention still further relates to a immunohistochemistry (IHC) kit for the detection of BAG3 protein in a biological sample, wherein said biological sample is preferably a tissue sample.
• Tissue samples can be biopsies, frozen tissues, paraffin embedded tissues.
• the IHC kit according to the present invention comprises BAG3- specific antibodies and reagents including probes needed for the staining.
• Said BAG3- specific antibodies can be mouse monoclonal antibodies AC-1, AC-2 and AC-3 and/or enzyme-linked antibodies for the detection able to recognize mouse immunoglobulins.
• the IHC kit advantageously allows to reveal BAG3 protein in 100% of pancreatic carcinoma tissue samples from patients that undergo pancreas resection and is expressed in most bladder carcinoma samples.
• BAG3 protein can be revealed with the kit for BAG3 detection by IHC also in normal pancreas tissue in Langerhans islets while other normal tissues result negative (such for example normal urocystis). BAG3 positivity can be also observed in synovial fibroblasts and inflammatory infiltrates in rheumatoid arthritis tissue samples .
• the BAG3 IHC kit allows the identification of the prognosis of PDAC patients. It was seen that the intensity of BAG3 expression identified by IHC, correlates with patients' survival. Therefore it can be used for both prognosis and for making a choice of therapy.
• a still further aspect of the invention is a kit for the detection of BAG3 gene expression in a biological sample comprising a set of amplification primers.
• amplification primers are suitable for the detection by quantitative real-time RT-PCR.
• fw SEQ ID NO. 3: CCGGCTGGCCCTTCTTCGTG;
• fw SEQ ID NO. 7: CCAGCCTCCCACGGACCTGA;
• the RT-PCR kit for bag3 mRNA detection in a biological sample allows to correlate the levels of bag3 gene expression with patients' survival and can be used for prognosis and for choice of therapy.
• said biological sample is a tissue sample.
• a still further aspect of the invention is represented by anti-BAG3 monoclonal antibodies, their fragments, and peptides corresponding to specific aminoacidic sequences of BAG3 protein that are able to block macrophage activation and can therefore be used for therapy of inflammatory, oncologic or other diseases involving macrophage activation. See in particular Figure 3 and Table I.
• This invention relates to the use of BAG3- specific mouse monoclonal antibodies AC-1, AC-2 and AC-3 or same modified as F(ab), F(ab')2, F(ab) or humanized; or peptides comprising sequences as follows:
• PEP4 NPSSMTDTPGNPAAP (SEQ ID NO. 14)
• Cardiomyocytes are known to release protective factors in mounting a response against stressful agents. Since stress- induced proteins, such as Hsp70, Hp27, Hsp90 and others, although exerting an intracellular activity, can also be secreted in response to stress, BAG3 release by cardiomyocytes was analyzed in stressful conditions. For this purpose, we analyzed the effect of serum deprivation- induced stress in cultured human primary cardiomyocytes or the rat cardiomyocyte cell line H9c2. As shown in Figure 1, we could detect BAG3 protein in the supernatants of cardiomyocytes exposed to serum deprivation for 16 h ( Figure 1A) .
• the human BAG 3 protein has the amino acid sequence according to SEQ ID NO: 15.
• J774 cells were incubated with 14 nM FITC-BAG3 protein and with 625 nM of BAG3 peptides (peptide 1, peptide 2, peptide 3, peptide 4 or scrambled peptide) or with 420 nM of
• F(ab')2 fragments from anti-BAG3 monoclonal and polyclonal antibodies mouse monoclonal AC1, AC2 and rabbit polyclonal TOS2).
• IgG were used as a negative control.
• BAG3-treated macrophages Furthermore, BAG3 induced the release of nitrite and interleukin
• BAG3-specific mouse monoclonal antibodies AC-1, AC-2 and AC-3 and/or others or same modified as F(ab), F(ab')2, F(ab) or humanized; or peptides comprising sequences PEP 1 to 4 and/or others are molecules able to bind and/or block soluble BAG3 effects to be used for therapy of inflammatory, oncologic or other diseases involving macrophage activation.
• BAG3 expression in PDAC by immunohistochemistry was developed an immunohistochemistry (IHC) kit, including our anti-BAG3 monoclonal antibodies and able to detect BAG3 protein by immunohistochemistry (IHC).
• IHC immunohistochemistry
• This kit revealed BAG3 expression in all the 346 (100%) PDAC biopsies that we analyzed.
• BAG3 staining revealed a moderate positivity of Langerhans islets, while normal pancreatic ducts and pancreatic acinar cells had no BAG3 expression. This was true in both normal pancreas and non-neoplastic pancreatic tissue adjacent to the tumor mass. BAG3 staining was observed predominantly in the cytoplasm of tumor cells.
• the intensity of staining of BAG3 was variable as was the number of positive cancer cells. Furthermore, Langerhans insulae were positive and constituted a good internal control of IHC ( Figure 4A) . Therefore our kit allows the detection of BAG3 protein in PDAC by IHC. Furthermore, it allows detecting BAG3 protein by IHC also in other tumour or normal tissues
• the survival analysis was performed in a cohort of 66 patients of which all the lesions examined were with resection margins free from tumor cells (R0) and only the 3.7% showed the presence of metastases to distant organs (Table II) .
• the first-line chemotherapy for treatment of pancreatic cancer is gemcitabine.
• BAG3 protein in response to therapy, we analyzed the effect of BAG3 down- modulation in human PDAC cells.
• Silencing of BAG3 enhanced cell apoptosis in response to the drug ( Figure 8) .
• bag3 primers that detect BAG3 expression by quantitative real-time RT-PCR.
• RT-PCR kit containing specific primers for bag3 mRNA detection and quantification.
• Control sera were obtained from healthy subjects and we measure BAG3 antibody titers in about five control subjects compared to each PDAC case. Furthermore, control sera came from subjects age matched with the respective PDAC case. This control sera matching was used for each time point.
• BAG3 antibodies concentrations were detected in a specific ELISA assay.
• BAG3 antibodies were significantly higher in cases at least 3-4 years pre-diagnosis in respect to control subjects.
• BAG3 antibodies were significantly higher in cases at least 3-4 years pre-diagnosis in respect to control subjects.
• HCMa Human Cardiac Myocytes-adult ) were purchased from Sciencell Research Laboratories (San Diego, CA) and grown in Cardiac Myocyte Medium (CMM, FBS 5%, Cardiac Myocyte Growth Supplement 1%, penicillin/ streptomycin solution 1%) (Sciencell Research Laboratories, San Diego, CA) . All experiments were performed on low- passage cell cultures.
• Embryonic rat cardiomyoblasts (line H9c2) was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10 % fetal bovine serum (FBS), 100 U/mL penicillin and 100 g/mL streptomycin.
• DMEM Dulbecco's Modified Eagle's Medium
• FBS fetal bovine serum
• J774A.1 murine monocyte macrophage cell line (ATCC, Manassas, VA, USA), was grown in DMEM supplemented with 10% fetal bovine serum (FBS), 25 mM HEPES, 2 mM glutamine, 100 u/mL penicillin and 100 g/mL streptomycin.
• pancreatic cancer cell lines (MIA PaCa-2, AsPC-1, PSN1, Capan-1 and PANC-1) were received from the American Type Culture Collection (ATCC; Manassas, VA) cell bank.
• MIA PaCa-2 cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) and supplemented with 10% FBS and 2.5% horse serum.
• AsPC-1 and PSN1 cells were grown in RPMI-1640 Medium supplemented with 10% FBS.
• Capan-1 were cultured in RPMI-1640 containing 20% FBS while PANC-1 were cultured in DMEM supplemented with 10% FBS.
• Dissociation of BAG3 antibodies in human sera Sera were diluted 1:40 with dissociation buffer (PBS with 1.5% BSA and 0.2 M glycine-acetate pH 2.5) to a 500 ⁇ final volume and incubated for 20 min at room temperature.
• the sera were then pipetted into the sample reservoir of Microcon centrifugal filter device, YM-100 (100,000 MW cut-off; Millipore, Billerica, MA, USA) and centrifuged at 14,100 rpm for 20 min at room temperature.
• the sample reservoir was then separated from the flow through, placed inverted into a second tube and centrifuged at 5, 000 rpm for 3 min at room temperature.
• the collected solution containing the antibody dissociated was adjusted to pH 7.0 with 1 M Tris buffer, pH 9.0.
• the retentate volume was reconstituted to the initial volume (500 ⁇ ) with dilution buffer ( PBS with 1.5% BSA and 0.1% Tween-20) .14
• the dissociated antibodies were diluted 1:200 in TBST containing 5% bovine serum albumin overnight at 4°C.
• Cells were harvested and lysed in a buffer containing 20 mM HEPES (pH 7.5), 150 mM NaCl, 0.1% Triton (TNN buffer) supplemented with a protease inhibitors cocktail (1 mM phenylmethylsulfonyl fluoride, 1 mg/ml pepstatin A, 2 mg/ml aprotinin) by 3 cycles of freezing and thawing. Soluble proteins were collected after a centrifugation at 10, 000 g for 15 min and their amount was determined by Bradford assay (Bio-Rad, Hercules, CA) . 25 g of total protein and serum samples (1:2 in PBS-T 0.05%) were run on 8% or 10% SDS-PAGE gels and electrophoretically transferred to nitrocellulose membrane.
• Nitrocellulose blots were blocked with 10% nonfat dry milk in TBST buffer (20 mM Tris-HCl pH 7.4, 500 mM NaCl, and 0.1% Tween 20) and incubated with primary antibodies in TBST containing 5% bovine serum albumin or 5% non-fat dry milk, overnight at 4°C. Immunoreactivity was detected by sequential incubation with horseradish peroxidase-conjugated secondary antibodies purchased from Pierce (Rockford, IL) and ECL detection reagents purchased from Amersham Life Sciences Inc. (Arlington Heights, IL, USA) .
• Protein bands were excised and gel pieces were subsequently washed with MilliQ Water and Acetonitrile and the proteins were digested in situ as described in Shevchenko protocol. Briefly, gel slices were reduced in 1,4- dithiothreitol (10 mM) and alkylated with iodoacetamide (50 mM) , then washed and rehydrated in trypsin solution (12 ng/ L) on ice for lh. After the addition of 30 ammonium bicarbonate
• Serum-free medium of H9c2 was cleared of cells and large debris by serial centrifugation at 4°C
• F(ab')2 fragments from anti-BAG3 monoclonal and polyclonal antibodies (mouse monoclonal AC1, AC2 and rabbit polyclonal TOS2) or F(ab')2 fragments from mouse IgG or F(ab')2 fragments from rabbit IgG in PBS containing 2% FBS+ 0.1 % NaN3 for 30 min on ice.
• IL6 was measured in supernatant of J774 A.l cells (5xl0 4 / in 96-well microplates) treated with LPS (10 ng/ml) or with rBAG3 (14 nM) or BSA (14 nM) for 10 or 20 hours in absence or presence of polymyxin B sulfate (5 g/ml) . After treatment 50 L of cell culture medium were collected and analyzed in triplicate with a mouse IL6 Kit
• rBAG3-FITC and BSA-FITC proteins were added in HCMa and J774 Al culture media with 0.1 % NaN 3 for 1 h.
• Coverslips were washed in lx PBS and fixed in 3.7% formaldehyde in lx PBS for 30 min at room temperature, and then incubated for 5 min with lx PBS 0.1M glycine. Following incubation with a 1:100 dilution of anti-P-integrin monoclonal antibody at 4 °C, coverslips were washed three times with lx PBS.
• Images were acquired in sequential scan mode by using the same acquisitions parameters (laser intensities, gain photomultipliers , pinhole aperture, objective 63X, zoom 2) when comparing experimental and control material.
• brightness and contrast of images were adjusted by taking care to leave a light cellular fluorescence background for visual appreciation of the lowest fluorescence intensity features and to help comparison among the different experimental groups.
• Final figures were assembled using Adobe Photoshop 7 and Adobe Illustrator 10. Leica Q9 Confocal Software and ImageJ were used for data analysis.
• NUNC Maxisorp 96 well ELISA plates were coated with recombinant BAG3 protein l g/ml (50 l/well) in PBS, pH 7 and incubated overnight at 4°C. Plates were washed 2 times with washing buffer (PBS + 0.05% Tween-20), and then blocked (150 ⁇ /well) for one hour at room temperature with 0.5% fish gelatin in PBS. Following blocking, the plates were washed 2 times with washing buffer and sera were diluted 1:70 with 0.5% fish gelatin in washing buffer and then applied (50 ⁇ /well) in triplicate and incubated at room temperature for two hour. The plates were then washed 6 times with washing buffer.
• washing buffer PBS + 0.05% Tween-20
• Anti-human IgG (H+L) antibody (Sigma Aldrich) was diluted 1:20,000 with 0.5% fish gelatin in washing buffer, added at 50 ⁇ /well and incubated at 4°C for 30 minutes. After incubation, the plates were washed 6 times, developed with TMB (50 ⁇ /well) (eBioscience ) , the reaction stopped with 4.5 M sulfuric acid (25 ⁇ /well) and the plates were analyzed spectrophotometrically at 450 nm.
• N0 2 ⁇ assay Nitrite content (N02 ⁇ ) a stable metabolite of NO released by cells in the culture supernatant, was measured in J774 A.l cells (5x104/ in 96-well microplates) treated with LPS (10 ng/ml) or with rBAG3 (7, 14 and 28 nM) or BSA (28 nM) for 24 hours in absence or presence of polymyxin B sulfate (Sigma-Aldrich, St. Louis, MO, USA) 5 g/ml. N02 ⁇ amounts were measured by Griess reaction.
• NUNC Maxisorp 96 well ELISA plates were coated with anti-BAG3 monoclonal antibody AC-1, AC-2 or AC-3 in PBS, pH 7 and incubated overnight at 4°C. Plates were washed 2 times with washing buffer (PBS + 0.05% Tween-20), and then blocked (150 ⁇ /well) for one hour at room temperature with 0.5% fish gelatin in PBS. Following blocking, the plates were washed 2 times with washing buffer and sera were diluted 1:70 with 0.5% fish gelatin in washing buffer and then applied (50 ⁇ /well) in triplicate and incubated at room temperature for two hour. The plates were then washed 6 times with washing buffer.
• washing buffer PBS + 0.05% Tween-20
• Anti-human IgG (H+L) antibody (Sigma Aldrich) was diluted 1:20,000 with 0.5% fish gelatin in washing buffer, added at 50 ⁇ /well and incubated at 4°C for 30 minutes. After incubation, the plates were washed 6 times, developed with TMB (50 ⁇ /well) (eBioscience ) , the reaction stopped with 4.5 M sulfuric acid (25 ⁇ /well) and the plates were analyzed spectrophotometrically at 450 nm.
• Immunohistochemistry protocol included: deparaffination in xylene, re-hydration through descending concentrations of alcohol up to pure water, non-enzymatic antigen retrieval in citrate buffer, pH 6.0, for 30 minutes at 95°C, and endogenous peroxidase quenching with H202 in methanol for 20 minutes. After rinsing with PBS, the samples were blocked with 5% normal horse serum in 0.1% PBS/BSA. To detect BAG3, samples were incubated for 1 hour at room temperature with BAG3 monoclonal antibody AC-1, AC-2 or AC-3 at the concentration of 3 microg/ml. After washing thoroughly with PBS, sections were incubated with a biotinylated secondary anti- mouse IgG for 20 minutes, then rinsed, incubated with avidin-biotin-complexes peroxidase
• RNA concentration and purity were validated by NanoDrop Spectrophotometer (Thermo Fisher, Waltham, MA, USA).
• RNA was reverse-transcribed using the High-Capacity cDNA Reverse Transcription Kit according to the manufacturer's instructions (Applied Biosystems, Applera, Foster City, CA, USA) .
• Quantitative real-time PCR assay was used to assess the differential expression of BAG3 in tumor tissue samples.
• Primers for the human bag3 gene were synthesized by Primm srl (Milano, Italy) (forward primer: (SEQ ID NO: 16) CCT GTT AGC TGT GGT TG; reverse primer: (SEQ ID NO: 17) AAC ATA CAG ATA TTC CTA TGG C) .

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