Our study also provides a novel explanation for unpredicted negative trial results and could be used for optimization of future combination therapies. Funding This work was supported from the Cancer Research UK Programme Grant (C2259/A16569) and by the Medical Research Council for the Councils Co-operative Awards in Science and Engineering (CASE). cellular proliferation and apoptosis (caspase 3/7 activity reduced by 1.4-fold, 95% confidence interval [CI] = 0.78 to 2.11, = .003) as opposed to synergistic effects observed with the irinotecan metabolite 7-Ethyl-10-hydroxycamptothecin (SN-38). Although both oxaliplatin and SN-38 produced ROS, only oxaliplatin-mediated apoptosis was ROS dependent. Production of ROS by oxaliplatin was secondary to STAT1-mediated transcriptional upregulation of DUOX2 (3.1-fold, 95% CI = 1.75 to 2.41, < .001). Inhibition of DUOX2 induction and p38 activation by cetuximab reduced oxaliplatin cytotoxicity. Conclusions: Inhibition of STAT1 and DUOX2-mediated ROS generation by cetuximab impairs p38-dependent apoptosis by oxaliplatin in preclinical models and may contribute to reduced efficacy in medical settings. Understanding the rationale for unpredicted trial results will inform improved rationales for combining EGFR inhibitors with chemotherapeutic providers in future restorative use. In view of the importance of the epidermal growth element receptor (EGFR) in the development and maintenance of human being cancers, there is considerable desire for inhibiting this pathway with monoclonal antibodies or small molecule inhibitors (1C4). Antibodies inhibiting EGFR, including cetuximab and panitumumab, have shown effectiveness in colorectal malignancy (CRC) either as monotherapy, or in combination with chemotherapy (5C8). Preclinical and medical studies of cetuximab or panitumumab with irinotecan-based chemotherapy have shown benefit in CRC (9C10). In contrast, despite some effectiveness for antibodies focusing on EGFR and oxaliplatin mixtures (11C12), other studies have suggested either no benefit or a negative connection. A randomized study using cetuximab in combination with oxaliplatin and fluoropyrimidines to treat CRC showed no benefit from addition of cetuximab (13). More recently, the randomized NEW EPOC study of oxaliplatin and 5-fluorouracil alone or combined with cetuximab shown decreased progression-free and general success with cetuximab (14). Cisplatin and oxaliplatin induce intra- and interstrand DNA cross-links, DNA-protein adducts (15C17), and generate development of reactive air types (ROS) and poisonous air metabolites, which trigger cytotoxic results by inducing DNA harm and apoptosis (18C21). Provided insufficient synergy Des and feasible antagonism of oxaliplatin coupled with cetuximab in CRC, we looked into potential systems of interaction. Strategies Reagents and Antibodies Cetuximab (5mg/mL) was extracted from Merck Serono KGaA (Darmstadt, Germany). EMD Serono (Boston, MA) supplied the MEK inhibitor pimasertib. SN-38, p38 inhibitor (SB202190), N-Acetyl-L-Cysteine (NAC), L-Ascorbic acidity, and MTT had been bought from Sigma-Aldrich. Reagents/antibodies useful for immunoblotting are detailed in the Supplementary Strategies (available on the web). Cell Lines and Lifestyle Circumstances Merck Serono (Darmstadt) supplied the SW48 cell range, and Bert Vogelstein (Johns Hopkins College or university) the DLD-1 isogenic KRAS wild-type cell range. Cells had been cultured in McCoys 5A customized mass media (Sigma-Aldrich), supplemented with 10% fetal bovine serum (Gibco), 2mM L-glutamine (Sigma), and 2mM penicillin-streptomycin (PAA). Cell lines had been authenticated in-may 2015 (LGC specifications). Immunoblotting Immunoblotting was performed as referred to (22). Detailed strategies are given in the Supplementary Components (obtainable online). ROS Recognition ROS levels had been detected using the cell-permeable substance H2DCFDA (Lifestyle Technology). Drug-treated cells or control cells had been washed double in PBS and incubated with PBS-H2DCFDA at 37 (1 M) for thirty minutes. Pursuing incubation using the ROS sign, cells had been cleaned in PBS double, collected and trypsinized. Samples were examined using a movement cytometer (CyAn ADP), and ROS was assessed as mean fluorescence strength. Results were examined using the Summit v4.3 software. Apoptosis and Cell Viability Dimension Apoptosis was assessed by Caspase 3/7 Glo assay and cell viability by Cell Titre Glo assay (Promega) based on the producers protocol. Drug Mixture Assays Ten thousand cells per well had been seeded within a 96-well dish (Corning) and drug-treated for 72 hours with cetuximab, oxaliplatin, SN-38, or their mixture; inhibition of proliferation was assessed by MTT assay. Synergy or antagonism had been motivated with Calcusyn software program using technique of Chou and Talalay VX-770 (Ivacaftor) (23). Medication arranging and dosing is certainly supplied in the Supplementary Components (obtainable online). Real-Time Polymerase String Reaction Oxidative Tension Arrays Real-time polymerase string response (RT-PCR) oxidative tension arrays (Qiagen).Practical cells (%) were measured with the Cell Titre Glo assay (Promega), and treated samples were normalized to neglected control (mean SD; n = 3). 2.11, = .003) instead of synergistic results observed using the irinotecan metabolite 7-Ethyl-10-hydroxycamptothecin (SN-38). Although both oxaliplatin and SN-38 created ROS, just oxaliplatin-mediated apoptosis was ROS reliant. Creation of ROS by oxaliplatin was supplementary to STAT1-mediated transcriptional upregulation of DUOX2 (3.1-fold, 95% CI = 1.75 to 2.41, < .001). Inhibition of DUOX2 induction and p38 activation by cetuximab decreased oxaliplatin cytotoxicity. Conclusions: Inhibition of STAT1 and DUOX2-mediated ROS era by cetuximab impairs p38-reliant apoptosis by oxaliplatin in preclinical versions and may donate to decreased efficacy in scientific settings. Understanding the explanation for unforeseen trial outcomes will inform improved rationales for merging EGFR inhibitors with chemotherapeutic agencies in future healing use. Because of the need for the epidermal development aspect receptor (EGFR) in the advancement and maintenance of individual cancers, there is certainly considerable fascination with inhibiting this pathway with monoclonal antibodies or small molecule inhibitors (1C4). Antibodies inhibiting EGFR, including cetuximab and panitumumab, have shown efficacy in colorectal cancer (CRC) either as monotherapy, or in combination with chemotherapy (5C8). Preclinical and clinical studies of cetuximab or panitumumab with irinotecan-based chemotherapy have shown benefit in CRC (9C10). In contrast, despite some efficacy for antibodies targeting EGFR and oxaliplatin combinations (11C12), other studies have suggested either no benefit or a negative interaction. A randomized study using cetuximab in combination with oxaliplatin and fluoropyrimidines to treat CRC showed no benefit from addition of cetuximab (13). More recently, the randomized NEW EPOC study of oxaliplatin and 5-fluorouracil alone or combined with cetuximab demonstrated reduced progression-free and overall survival with cetuximab (14). Cisplatin and oxaliplatin induce intra- and interstrand DNA cross-links, DNA-protein adducts (15C17), and generate formation of reactive oxygen species (ROS) and toxic oxygen metabolites, which cause cytotoxic effects by inducing DNA damage and apoptosis (18C21). Given lack of synergy and possible antagonism of oxaliplatin combined with cetuximab in CRC, we investigated potential mechanisms of interaction. Methods Reagents and Antibodies Cetuximab (5mg/mL) was obtained from Merck Serono KGaA (Darmstadt, Germany). EMD Serono (Boston, MA) provided the MEK inhibitor pimasertib. SN-38, p38 inhibitor (SB202190), N-Acetyl-L-Cysteine (NAC), L-Ascorbic acid, and MTT were purchased from Sigma-Aldrich. Reagents/antibodies used for immunoblotting are listed in the Supplementary Methods (available online). Cell Lines and Culture Conditions Merck Serono (Darmstadt) provided the SW48 cell line, and Bert Vogelstein (Johns Hopkins University) the DLD-1 isogenic KRAS wild-type cell line. Cells were cultured in McCoys 5A modified media (Sigma-Aldrich), supplemented with 10% fetal bovine serum (Gibco), 2mM L-glutamine (Sigma), and 2mM penicillin-streptomycin (PAA). Cell lines were authenticated in May 2015 (LGC standards). Immunoblotting Immunoblotting was performed as described (22). Detailed methods are provided in the Supplementary Materials (available online). ROS Detection ROS levels were detected with the cell-permeable compound H2DCFDA (Life Technologies). Drug-treated cells or control cells were washed twice in PBS and then incubated with PBS-H2DCFDA at 37 (1 M) for 30 minutes. Following incubation with the ROS indicator, cells were washed twice in PBS, trypsinized and collected. Samples were analyzed using a flow cytometer (CyAn ADP), and ROS was measured as mean fluorescence intensity. Results were analyzed with the Summit v4.3 software. Apoptosis and Cell Viability Measurement Apoptosis was measured by Caspase 3/7 Glo assay and cell viability by Cell Titre Glo assay (Promega) according to the manufacturers protocol. Drug Combination Assays Ten thousand cells per well were seeded in a 96-well plate (Corning) and drug-treated for 72 hours with cetuximab, oxaliplatin, SN-38, or their combination; inhibition of proliferation was measured by MTT assay. Synergy or antagonism were determined with Calcusyn software using methodology of Chou and Talalay (23). Drug scheduling and dosing is provided in the Supplementary Materials (available online). Real-Time Polymerase Chain Reaction Oxidative Stress Arrays Real-time polymerase chain reaction (RT-PCR) oxidative stress arrays (Qiagen) were used to measure RNA expression of stress-related genes following cetuximab and oxaliplatin treatment. Additional details are provided in the Supplementary Materials (available online). Chromatin Immunoprecipitation SW48 cells were treated with oxaliplatin (50 M), and proteins were cross-linked with 1% formaldehyde (Sigma-Aldrich) for 10 minutes at ambient temperature. Cells were lysed and chromatin extracted, and STAT-1 binding to the DUOX2 promoter was assessed by RT-PCR. Detailed assay protocol is described in the Supplementary Materials.F) Representative images at 40x magnification resulting from ISH and IHC analysis (scale bars = 50 M). and oxaliplatin exhibited antagonistic effects on cellular proliferation and apoptosis (caspase 3/7 activity reduced by 1.4-fold, 95% confidence interval [CI] = 0.78 to 2.11, = .003) as opposed to synergistic effects observed with the irinotecan metabolite 7-Ethyl-10-hydroxycamptothecin (SN-38). Although both oxaliplatin and SN-38 produced ROS, only oxaliplatin-mediated apoptosis was ROS dependent. Production of ROS by oxaliplatin was secondary to STAT1-mediated transcriptional upregulation of DUOX2 (3.1-fold, 95% CI = 1.75 to 2.41, < .001). Inhibition of DUOX2 induction and p38 activation by cetuximab reduced oxaliplatin cytotoxicity. Conclusions: Inhibition of STAT1 and DUOX2-mediated ROS generation by cetuximab impairs p38-dependent apoptosis by oxaliplatin in preclinical models and may contribute to reduced efficacy in clinical settings. Understanding the rationale for unexpected trial results will inform improved rationales for combining EGFR inhibitors with chemotherapeutic agents in future therapeutic use. In view of the importance of the epidermal growth factor receptor (EGFR) in the development and maintenance of human cancers, there is considerable interest in inhibiting this pathway with monoclonal antibodies or small molecule inhibitors (1C4). Antibodies inhibiting EGFR, including cetuximab and panitumumab, have shown efficacy in colorectal cancer (CRC) either as monotherapy, or in combination with chemotherapy (5C8). Preclinical and clinical studies of cetuximab or panitumumab with irinotecan-based chemotherapy have shown advantage in CRC (9C10). On the other hand, despite some efficiency for antibodies concentrating on EGFR and oxaliplatin combos (11C12), other research have recommended either no advantage or a poor connections. A randomized research using cetuximab in conjunction with oxaliplatin and fluoropyrimidines to take care of CRC demonstrated no reap the benefits of addition of cetuximab (13). Recently, the randomized NEW EPOC research of oxaliplatin and 5-fluorouracil alone or coupled with cetuximab showed decreased progression-free and general success with cetuximab (14). Cisplatin and oxaliplatin induce intra- and interstrand DNA cross-links, DNA-protein adducts (15C17), and generate development of reactive air types (ROS) and dangerous air metabolites, which trigger cytotoxic results by inducing DNA harm and apoptosis (18C21). Provided insufficient synergy and feasible antagonism of oxaliplatin coupled with cetuximab in CRC, we looked into potential systems of interaction. Strategies Reagents and Antibodies Cetuximab (5mg/mL) was extracted from Merck Serono KGaA (Darmstadt, Germany). EMD Serono (Boston, MA) supplied the MEK inhibitor pimasertib. SN-38, p38 inhibitor (SB202190), N-Acetyl-L-Cysteine (NAC), L-Ascorbic acidity, and MTT had been bought from Sigma-Aldrich. Reagents/antibodies employed for immunoblotting are shown in the Supplementary Strategies (available on the web). Cell Lines and Lifestyle Circumstances Merck Serono (Darmstadt) supplied the SW48 cell series, and Bert Vogelstein (Johns VX-770 (Ivacaftor) Hopkins School) the DLD-1 isogenic KRAS wild-type cell series. Cells had been cultured in McCoys 5A improved mass media (Sigma-Aldrich), supplemented with 10% fetal bovine serum (Gibco), 2mM L-glutamine (Sigma), and 2mM penicillin-streptomycin (PAA). Cell lines had been authenticated in-may 2015 (LGC criteria). Immunoblotting Immunoblotting was performed as defined (22). Detailed strategies are given in the Supplementary Components (obtainable online). ROS Recognition ROS levels had been detected using the cell-permeable substance H2DCFDA (Lifestyle Technology). Drug-treated cells or control cells had been washed double in PBS and incubated with PBS-H2DCFDA at 37 (1 M) for thirty minutes. Pursuing incubation using the ROS signal, cells were cleaned double in PBS, trypsinized and gathered. Samples were examined using a stream cytometer (CyAn ADP), and ROS was assessed as mean fluorescence strength. Results were examined using the Summit v4.3 software. Apoptosis and Cell Viability Dimension Apoptosis was assessed by Caspase 3/7 Glo assay and cell viability by Cell Titre Glo assay (Promega) based on the producers protocol. Drug Mixture Assays Ten thousand cells per well had been seeded within a 96-well dish (Corning) and drug-treated for 72 hours with cetuximab, oxaliplatin, SN-38, or their mixture; inhibition of proliferation was assessed by MTT assay. Antagonism or Synergy were determined with Calcusyn software program using.E and F) Apoptosis was measured by caspase 3/7 activity. utilized. Statistical tests had been two-sided. Outcomes: Cetuximab and oxaliplatin exhibited antagonistic results on mobile proliferation and apoptosis (caspase 3/7 activity decreased by 1.4-fold, 95% confidence interval [CI] = 0.78 to 2.11, = .003) instead of synergistic results observed using the irinotecan metabolite 7-Ethyl-10-hydroxycamptothecin (SN-38). Although both oxaliplatin and SN-38 created ROS, just oxaliplatin-mediated apoptosis was ROS reliant. Creation of ROS by oxaliplatin was supplementary to STAT1-mediated transcriptional upregulation of DUOX2 (3.1-fold, 95% CI = 1.75 to 2.41, < .001). Inhibition of DUOX2 induction and p38 activation by cetuximab decreased oxaliplatin cytotoxicity. Conclusions: Inhibition of STAT1 and DUOX2-mediated ROS era by cetuximab impairs p38-reliant apoptosis by oxaliplatin in preclinical versions and may donate to decreased efficacy in scientific settings. Understanding the explanation for unforeseen trial outcomes will inform improved rationales for merging EGFR inhibitors with chemotherapeutic realtors in future healing use. Because of the need for the epidermal development aspect receptor (EGFR) in the advancement and maintenance of individual cancers, there is certainly considerable curiosity about inhibiting this pathway with monoclonal antibodies or little molecule inhibitors (1C4). Antibodies inhibiting EGFR, including cetuximab and panitumumab, show efficiency in colorectal cancers (CRC) either as monotherapy, or in conjunction with chemotherapy (5C8). Preclinical and scientific research of cetuximab or panitumumab with irinotecan-based chemotherapy show advantage in CRC (9C10). On the other hand, despite some efficiency for antibodies concentrating on EGFR and oxaliplatin combos (11C12), other research have suggested either no benefit or a negative conversation. A randomized study using cetuximab in combination with oxaliplatin and fluoropyrimidines to treat CRC showed no benefit from addition of cetuximab (13). More recently, the randomized NEW EPOC study of oxaliplatin and 5-fluorouracil alone or combined with cetuximab exhibited reduced progression-free and overall survival with cetuximab (14). Cisplatin and oxaliplatin induce intra- and interstrand DNA cross-links, DNA-protein adducts (15C17), and generate formation of reactive oxygen species (ROS) and harmful oxygen metabolites, which cause cytotoxic effects by inducing DNA damage and apoptosis (18C21). Given lack of synergy and possible antagonism of oxaliplatin combined with cetuximab in CRC, we investigated potential mechanisms of interaction. Methods Reagents and Antibodies Cetuximab (5mg/mL) was obtained from Merck Serono KGaA (Darmstadt, Germany). EMD Serono (Boston, MA) provided the MEK inhibitor pimasertib. SN-38, p38 inhibitor (SB202190), N-Acetyl-L-Cysteine (NAC), L-Ascorbic acid, and MTT were purchased from Sigma-Aldrich. Reagents/antibodies utilized for immunoblotting are outlined in the Supplementary Methods (available online). Cell Lines and Culture Conditions Merck Serono (Darmstadt) provided the SW48 cell collection, and Bert Vogelstein (Johns Hopkins University or college) the DLD-1 isogenic KRAS wild-type cell collection. Cells were cultured in McCoys 5A altered media (Sigma-Aldrich), supplemented with 10% fetal bovine serum (Gibco), 2mM L-glutamine (Sigma), and 2mM penicillin-streptomycin (PAA). Cell lines were authenticated in May 2015 (LGC requirements). Immunoblotting Immunoblotting was performed as explained (22). Detailed methods are provided in the Supplementary Materials (available online). ROS Detection ROS levels were detected with the cell-permeable compound H2DCFDA (Life Technologies). Drug-treated cells or control cells were washed twice in PBS and then incubated with PBS-H2DCFDA at 37 (1 M) for 30 minutes. Following incubation with the ROS indication, cells were washed twice in PBS, trypsinized and collected. Samples were analyzed using a circulation cytometer (CyAn ADP), and ROS was measured as mean fluorescence intensity. Results were analyzed with the Summit v4.3 software. Apoptosis and Cell Viability Measurement Apoptosis was measured by Caspase 3/7 Glo assay and cell viability by Cell Titre Glo assay (Promega) according to the manufacturers protocol. Drug Combination Assays Ten thousand cells per well were seeded in a 96-well plate (Corning) and drug-treated for 72 hours with cetuximab, oxaliplatin, SN-38, or their combination; inhibition of proliferation was measured by MTT assay. Synergy or antagonism were decided with Calcusyn software using methodology of Chou and Talalay (23). Drug scheduling and dosing is usually provided in the Supplementary Materials (available online). Real-Time Polymerase Chain Reaction Oxidative Stress Arrays Real-time polymerase chain reaction (RT-PCR) oxidative.Correction for multiple comparisons was applied for scrambled vs scrambled-oxaliplatin, scrambled vs STAT1 siRNA, scrambled vs scrambled-oxaliplatin, scrambled-oxaliplatin vs STAT1 siRNA, and scrambled-oxaliplatin vs STAT1 siRNA-oxaliplatin. Furthermore, levels of P-STAT1 and cleaved PARP produced by oxaliplatin were reduced by STAT1 knockdown in SW48 and DLD-1 cells (Figure 4E). [saline]; n = 3 mice per treatment group) were used. Statistical assessments were two-sided. Results: Cetuximab and oxaliplatin exhibited antagonistic effects on cellular proliferation and apoptosis (caspase 3/7 activity reduced by 1.4-fold, 95% confidence interval [CI] = 0.78 to 2.11, = .003) as opposed to synergistic effects observed with the irinotecan metabolite 7-Ethyl-10-hydroxycamptothecin (SN-38). Although both oxaliplatin and SN-38 produced ROS, only oxaliplatin-mediated apoptosis was ROS dependent. Production of ROS by oxaliplatin was secondary to STAT1-mediated transcriptional upregulation of DUOX2 (3.1-fold, 95% CI = 1.75 to 2.41, < .001). Inhibition of DUOX2 induction and p38 activation by cetuximab reduced oxaliplatin cytotoxicity. Conclusions: Inhibition of STAT1 and DUOX2-mediated ROS generation by cetuximab impairs p38-dependent apoptosis by VX-770 (Ivacaftor) oxaliplatin in preclinical models and may contribute to reduced efficacy in clinical settings. Understanding the rationale for unexpected trial results will inform improved rationales for combining EGFR inhibitors with chemotherapeutic brokers in future therapeutic use. In view of the importance of the epidermal growth factor receptor (EGFR) in the development and maintenance of human cancers, there is considerable interest in inhibiting this pathway with monoclonal antibodies or small molecule inhibitors (1C4). Antibodies inhibiting EGFR, including cetuximab and panitumumab, have shown efficacy in colorectal cancer (CRC) either as monotherapy, or in combination with chemotherapy (5C8). Preclinical and clinical studies of cetuximab or panitumumab with irinotecan-based chemotherapy have shown benefit in CRC (9C10). In contrast, despite some efficacy for antibodies targeting EGFR and oxaliplatin combinations (11C12), other studies have suggested either no benefit or a negative interaction. A randomized study using cetuximab in combination with oxaliplatin and fluoropyrimidines to treat CRC showed no benefit from addition of cetuximab (13). More recently, the randomized NEW EPOC study of oxaliplatin and 5-fluorouracil alone or combined with cetuximab demonstrated reduced progression-free and overall survival with cetuximab (14). Cisplatin and oxaliplatin induce intra- and interstrand DNA cross-links, DNA-protein adducts (15C17), and generate formation of reactive oxygen species (ROS) and toxic oxygen metabolites, which cause cytotoxic effects by inducing DNA damage and apoptosis (18C21). Given lack of synergy and possible antagonism of oxaliplatin combined with cetuximab in CRC, we investigated potential mechanisms of interaction. Methods Reagents and Antibodies Cetuximab (5mg/mL) was obtained from Merck Serono KGaA (Darmstadt, Germany). EMD Serono (Boston, MA) provided the MEK inhibitor pimasertib. SN-38, p38 inhibitor (SB202190), N-Acetyl-L-Cysteine (NAC), L-Ascorbic acid, and MTT were purchased from Sigma-Aldrich. Reagents/antibodies used for immunoblotting are listed in the Supplementary Methods (available online). Cell Lines and Culture Conditions Merck Serono (Darmstadt) provided the SW48 cell line, and Bert Vogelstein (Johns Hopkins University) the DLD-1 isogenic KRAS wild-type cell line. Cells were cultured in McCoys 5A modified media (Sigma-Aldrich), supplemented with 10% fetal bovine serum (Gibco), 2mM L-glutamine (Sigma), and 2mM penicillin-streptomycin (PAA). Cell lines were authenticated in May 2015 (LGC standards). Immunoblotting Immunoblotting was performed as described (22). Detailed methods are provided in the Supplementary Materials (available online). ROS Detection ROS levels were detected with the cell-permeable compound H2DCFDA (Life Technologies). Drug-treated cells or control cells were washed twice in PBS and then incubated with PBS-H2DCFDA at 37 (1 M) for 30 minutes. Following incubation with the ROS indicator, cells were washed twice in PBS, trypsinized and collected. Samples were analyzed using a flow cytometer (CyAn ADP), and ROS was measured as mean fluorescence intensity. Results were analyzed with the Summit v4.3 software. Apoptosis and Cell Viability Measurement Apoptosis was measured by Caspase 3/7 Glo assay and cell viability by Cell Titre Glo assay (Promega) according to the manufacturers protocol. Drug Combination Assays Ten thousand cells per well were seeded in a 96-well plate (Corning) and drug-treated for 72 hours with cetuximab, oxaliplatin, SN-38, or their combination; inhibition of proliferation was measured by MTT assay. Synergy or antagonism were determined with Calcusyn software using methodology of Chou and Talalay (23). Drug scheduling and dosing is provided in the Supplementary Materials (available online). Real-Time Polymerase Chain Reaction Oxidative Stress Arrays Real-time polymerase chain reaction (RT-PCR) oxidative stress arrays (Qiagen) were used to measure RNA expression of stress-related genes following cetuximab and oxaliplatin treatment. Additional details are provided in the Supplementary Materials (available online). Chromatin Immunoprecipitation SW48 cells were treated with oxaliplatin (50 M), and proteins were cross-linked with 1% formaldehyde (Sigma-Aldrich) for 10 minutes at ambient temperature. Cells were lysed and chromatin extracted, and STAT-1 binding to the DUOX2 promoter was assessed by RT-PCR. Detailed assay protocol is definitely explained in the Supplementary Materials (available on-line). Xenografts All in vivo experiments were performed according to the Animal Research.