BMDCs isolated from wild-type (WT) or BLTR1-deficient (KO) mice were stimulated with HMGB1 for 10 days, and then cellular expressions of CD11b/Mac-1 were determined by circulation cytometry. direct effects on MMD in control and 5-LO-deficient BMDCs, MMD attenuation by HMGB1 in 5-LO-deficient BMDCs was significantly reversed by exogenous LTB4, but not in BLTR1-deficient BMDCs, suggesting that LTB4/BLTR1-mediated priming of monocytes is usually a prerequisite of HMGB1-induced MMD. transfection Small interfering RNA (siRNA) for BLTR1 and scrambled siRNA (unfavorable control) were designed and synthesized using a SilencerTM siRNA construction kit purchased from Bioneer. The sequences of BLTR1 siRNA and scrambled siRNA were 5-GAUCUGCGCUCCGAACUAUdTdT-3 and 5-AUAGUUCGGAGCGCAGAUCdTdT-3, respectively. For siRNA transfection, cells were seeded and transfected with BLTR1 siRNA using Lipofectamine 2000 (Invitrogen, NY, USA) according to the manufacturer’s protocol. Transfection efficiencies were monitored using a fluorescent oligonucleotide (BLOCK-iT Fluorescent Oligo; Invitrogen) and estimated to be between 80 and 90%. Immunofluorescence analysis Wire-injured femoral arteries were harvested and serial paraffin sections (4 m) of femoral arteries were incubated with mouse-anti -SMA (1:400) and rabbit-anti CD36 (1:200) antibodies. Alexa488-conjugated IgG and Alexa594-conjugated IgG (Abcam) were used to detect immunofluorescence signals for -SMA and CD36, respectively. After nuclei were visualized by staining with 0.1 g/ml diamidino-2-phenylindole (DAPI), slides were mounted in Vectashield. Fluorescence images were visualized by scanning confocal microscopy (LSM 510, Carl Zeiss, Oberkochen, Germany), and analyzed by National Institutes of Health (NIH) image software (Image J, NIH, USA). Transplantation of bone marrow-derived monocytes BMDCs were harvested from your femurs and tibiae of mice (7 wks, male), which had been euthanized by carbon dioxide insufflation and cervical dislocation, and bone marrow-derived monocytes (BMDMs, CD11b-positive cells) were then separated using AS-605240 MACS technology (Miltenyi, Bergisch Gladbach, GER) using a standard procedure. BMDCs were then stained with fluorochrome-labeled monoclonal anti-CD11b, sorted using a BD ARIAIII cell sorter (Becton Dickinson, San Jose, CA, USA), washed, and resuspended at 1 107 cells/ml. Recipient BLTR1-deficient mice were administered 1 107 BMDMs per mouse by tail vein injection. The expressions of BLTR1 mRNA and protein in peripheral blood monocytes (PBMCs) isolated from three groups of BMDMs-transplanted mice (WTWT mice, WT monocytes into WT mice; KOKO mice, BLTR1-deficient monocytes into BLTR1-deficient mice; and WTKO mice, WT monocytes into BLTR1-deficient mice) were determined by Real Time PCR and immunocytochemistry, respectively. Statistical analysis Results were expressed as means SEMs. One-way analysis of variance (ANOVA) followed by Turkey’s multiple comparison test was used to determine the significance of differences. Statistical significance was accepted for values < 0.05. Results A role for 5-LO in MMD induced by HMGB1 The effects of HMGB1 around the expression of 5-LO mRNA and protein in BMDCs were decided using semi-quantitative RT-PCR and Western blot analysis. In previous studies, HMGB1 were secreted to 10C100 ng/ml physiologically or pathologically (27, 28). Thus, BMDCs were treated with HMGB1 at concentrations of 100 ng/ml in our study. As shown in Figure ?Physique1A,1A, HMGB1 at concentration of 100 ng/ml increased the mRNA and protein expression of 5-LO in a time-dependent manner in BMDCs and THP-1 cells (Supplementary Physique 1), which were attenuated by inhibition of various receptors for HMGB1 (Supplementary Shape AS-605240 2). To look for the practical part of 5-LO improved in HMGB1-activated cells, LTB4 creation in HMGB1-treated cells was assessed using ELISA. As demonstrated in Figure ?Shape1B,1B, LTB4 creation in HMGB1-stimulated cells was gradually increased up to 24 h (approximately 10 ng/107 cells), suggesting the participation of 5-LO-derived LTs in MMD induction by HMGB1. Open up in another window Shape 1 Part of 5-LO in monocytes on monocyte-to-macrophage differentiation (MMD) induced by HMGB1. (A) Time-courses from the expressions of 5-LO mRNA and proteins in BMDCs activated with HMGB1 (100 ng/ml) had been established using RT-PCR and Traditional western blot, respectively. Bottom level: Blot densities had been quantified and shown as the means SEMs of 6C7 3rd party tests. *< 0.05; **< 0.01 vs. related worth at 0 h. (B) Time-course of LTB4 creation in automobile- or HMGB1 (100 ng/ml)-activated BMDCs as dependant on ELISA. Bottom level: Color indicators had been quantified, and data had been presented as.To look for the functional part of 5-LO increased in HMGB1-stimulated cells, LTB4 creation in HMGB1-treated cells was measured using ELISA. a 5-LO inhibitor aswell as with 5-LO-deficient BMDCs. Of varied leukotriene receptor inhibitors analyzed, including leukotriene B4 receptors (BLTRs) and cysteinyl leukotriene receptors (cysLTRs), the BLTR1 inhibitor ("type":"entrez-nucleotide","attrs":"text":"U75302","term_id":"1857248"U75302) specifically suppressed MMD induction by HMGB1. The need for BLTR1 in HMGB1-induced MMD was also seen in BMDCs isolated from BLTR1-deficient BMDCs and mice transfected with BLTR1 siRNA. Although leukotriene B4 (LTB4) got minimal direct results on MMD in charge and 5-LO-deficient BMDCs, MMD attenuation by HMGB1 in 5-LO-deficient BMDCs was considerably reversed by exogenous LTB4, however, not in BLTR1-lacking BMDCs, recommending that LTB4/BLTR1-mediated priming of monocytes can be a prerequisite of HMGB1-induced MMD. transfection Little interfering RNA (siRNA) for BLTR1 and scrambled siRNA (adverse control) had been designed and synthesized utilizing a SilencerTM siRNA building kit bought from Bioneer. The sequences of BLTR1 siRNA and scrambled siRNA had been 5-GAUCUGCGCUCCGAACUAUdTdT-3 and 5-AUAGUUCGGAGCGCAGAUCdTdT-3, respectively. For siRNA transfection, cells had been seeded and transfected with BLTR1 siRNA using Lipofectamine 2000 (Invitrogen, NY, USA) based on the manufacturer's process. Transfection efficiencies had been monitored utilizing a fluorescent oligonucleotide (BLOCK-iT Fluorescent Oligo; Invitrogen) and estimated to become between 80 and 90%. Immunofluorescence evaluation Wire-injured femoral arteries had been gathered and serial paraffin areas (4 m) of femoral arteries had been incubated with mouse-anti -SMA (1:400) and rabbit-anti Compact disc36 (1:200) antibodies. Alexa488-conjugated IgG and Alexa594-conjugated IgG (Abcam) had been utilized to detect immunofluorescence indicators for -SMA and Compact disc36, respectively. After nuclei had been visualized by staining with 0.1 g/ml diamidino-2-phenylindole (DAPI), slides had been mounted in Vectashield. Fluorescence pictures had been visualized by checking confocal microscopy (LSM 510, Carl Zeiss, Oberkochen, Germany), and analyzed by Country wide Institutes of Wellness (NIH) image software program (Picture J, NIH, USA). Transplantation of bone tissue marrow-derived monocytes BMDCs had been harvested through the femurs and tibiae of mice (7 wks, male), which have been euthanized by skin tightening and insufflation and cervical dislocation, and bone AS-605240 tissue marrow-derived monocytes (BMDMs, Compact disc11b-positive cells) had been after that separated using MACS technology (Miltenyi, Bergisch Gladbach, GER) utilizing a regular procedure. BMDCs had been after that stained with fluorochrome-labeled monoclonal anti-CD11b, sorted utilizing a BD ARIAIII cell sorter (Becton Dickinson, San Jose, CA, USA), cleaned, and resuspended at 1 107 cells/ml. Receiver BLTR1-lacking mice were given 1 107 BMDMs per mouse by tail vein shot. The expressions of BLTR1 mRNA and proteins in peripheral bloodstream monocytes (PBMCs) isolated from three sets of BMDMs-transplanted mice (WTWT mice, WT monocytes into WT mice; KOKO mice, BLTR1-lacking monocytes into BLTR1-lacking mice; and WTKO mice, WT monocytes into BLTR1-deficient mice) had been determined by REAL-TIME PCR and immunocytochemistry, respectively. Statistical evaluation Results were indicated as means SEMs. One-way analysis of variance (ANOVA) accompanied by Turkey's multiple assessment test was utilized to look for the significance of variations. Statistical significance was approved for ideals < 0.05. Outcomes A job for 5-LO in MMD induced by HMGB1 The consequences of HMGB1 for the manifestation of 5-LO mRNA and proteins in BMDCs had been established using semi-quantitative RT-PCR and Traditional western blot evaluation. In previous research, HMGB1 had been secreted to 10C100 ng/ml physiologically or pathologically (27, 28). Therefore, BMDCs had been treated with HMGB1 at concentrations of 100 ng/ml inside our research. As demonstrated in Figure ?Shape1A,1A, HMGB1 at focus of 100 ng/ml increased the mRNA and proteins manifestation of 5-LO inside a time-dependent way in BMDCs and THP-1 cells (Supplementary Shape 1), that have been attenuated by inhibition of varied receptors for HMGB1 (Supplementary Shape 2). To look for the practical part of 5-LO improved in HMGB1-activated cells, LTB4 creation in HMGB1-treated cells was assessed using ELISA. As demonstrated in Figure ?Shape1B,1B, LTB4 creation in HMGB1-stimulated cells AS-605240 was gradually increased up to 24 h (approximately 10 ng/107 cells), suggesting the participation of 5-LO-derived LTs in MMD induction by HMGB1. Open up in another window Shape 1 Part of 5-LO in monocytes on monocyte-to-macrophage differentiation (MMD) induced by HMGB1. (A) Time-courses from the expressions of 5-LO mRNA and proteins in BMDCs activated with HMGB1 (100 ng/ml) had been established using RT-PCR and Traditional western blot, respectively..When BLTR1 mRNA amounts in PBMCs isolated through the three sets of BMDM-transplanted mice (WTWT, WT monocytes into WT mice; WTKO, WT monocytes into BLTR1-lacking mice; and KOKO, BLTR1-deficient monocytes into BLTR1-deficient mice) had been determined by REAL-TIME PCR, a rise in BLTR1 mRNA amounts in the monocytes of receiver mice was detected at 1 and 5 wks after adoptive transplantation (Figure ?(Figure6A).6A). inhibitors examined, which included leukotriene B4 receptors (BLTRs) and cysteinyl Lyl-1 antibody leukotriene receptors (cysLTRs), the BLTR1 inhibitor (“type”:”entrez-nucleotide”,”attrs”:”text”:”U75302″,”term_id”:”1857248″U75302) exclusively suppressed MMD induction by HMGB1. The importance of BLTR1 in HMGB1-induced MMD was also observed in BMDCs isolated from BLTR1-deficient mice and BMDCs transfected with BLTR1 siRNA. Although leukotriene B4 (LTB4) had minimal direct effects on MMD in control and 5-LO-deficient BMDCs, MMD attenuation by HMGB1 in 5-LO-deficient BMDCs was significantly reversed by exogenous LTB4, but not in BLTR1-deficient BMDCs, suggesting that LTB4/BLTR1-mediated priming of monocytes is a prerequisite of HMGB1-induced MMD. transfection Small interfering RNA (siRNA) for BLTR1 and scrambled siRNA (negative control) were designed and synthesized using a SilencerTM siRNA construction kit purchased from Bioneer. The sequences of BLTR1 siRNA and scrambled siRNA were 5-GAUCUGCGCUCCGAACUAUdTdT-3 and 5-AUAGUUCGGAGCGCAGAUCdTdT-3, respectively. For siRNA transfection, cells were seeded and transfected with BLTR1 siRNA using Lipofectamine 2000 (Invitrogen, NY, USA) according to the manufacturer’s protocol. Transfection efficiencies were monitored using a fluorescent oligonucleotide (BLOCK-iT Fluorescent Oligo; Invitrogen) and estimated to be between 80 and 90%. Immunofluorescence analysis Wire-injured femoral arteries were harvested and serial paraffin sections (4 m) of femoral arteries were incubated with mouse-anti -SMA (1:400) and rabbit-anti CD36 (1:200) antibodies. Alexa488-conjugated IgG and Alexa594-conjugated IgG (Abcam) were used to detect immunofluorescence signals for -SMA and CD36, respectively. After nuclei were visualized by staining with 0.1 g/ml diamidino-2-phenylindole (DAPI), slides were mounted in Vectashield. Fluorescence images were visualized by scanning confocal microscopy (LSM 510, Carl Zeiss, Oberkochen, Germany), and analyzed by National Institutes of Health (NIH) image software (Image J, NIH, USA). Transplantation of bone marrow-derived monocytes BMDCs were harvested from the femurs and tibiae of mice (7 wks, male), which had been euthanized by carbon dioxide insufflation and cervical dislocation, and bone marrow-derived monocytes (BMDMs, CD11b-positive cells) were then separated using MACS technology (Miltenyi, Bergisch Gladbach, GER) using a standard procedure. BMDCs were then stained with fluorochrome-labeled monoclonal anti-CD11b, sorted using a BD ARIAIII cell sorter (Becton Dickinson, San Jose, CA, USA), washed, and resuspended at 1 107 cells/ml. Recipient BLTR1-deficient mice were administered 1 107 BMDMs per mouse by tail vein injection. The expressions of BLTR1 mRNA and protein in peripheral blood monocytes (PBMCs) isolated from three groups of BMDMs-transplanted mice (WTWT mice, WT monocytes into WT mice; KOKO mice, BLTR1-deficient monocytes into BLTR1-deficient mice; and WTKO mice, WT monocytes into BLTR1-deficient mice) were determined by Real Time PCR and immunocytochemistry, respectively. Statistical analysis Results were expressed as means SEMs. One-way analysis of variance (ANOVA) followed by Turkey’s multiple comparison test was used to determine the significance of differences. Statistical significance was accepted for values < 0.05. Results A role for 5-LO in MMD induced by HMGB1 The effects of HMGB1 on the expression of 5-LO mRNA and protein in BMDCs were determined using semi-quantitative RT-PCR and Western blot analysis. In previous studies, HMGB1 were secreted to 10C100 ng/ml physiologically or pathologically (27, 28). Thus, BMDCs were treated with HMGB1 at concentrations of 100 ng/ml in our study. As shown in Figure ?Figure1A,1A, HMGB1 at concentration of 100 ng/ml increased the mRNA and protein expression of 5-LO in a time-dependent manner in BMDCs and THP-1 cells (Supplementary Figure 1), which were attenuated by inhibition of various receptors for HMGB1 (Supplementary Figure 2). To determine the functional role of 5-LO increased in HMGB1-stimulated cells, LTB4 production in HMGB1-treated cells was measured using ELISA. As shown in Figure ?Figure1B,1B, LTB4 production in HMGB1-stimulated cells was gradually increased up to 24 h (approximately 10 ng/107 cells), suggesting the potential involvement of 5-LO-derived LTs in MMD induction by HMGB1. Open in a separate window Figure 1.Reportedly, HMGB1 is known to have pro-inflammatory cytokine-like activity, promote chemotaxis, and stimulate cellular migration and growth (35). isolated from BLTR1-deficient mice and BMDCs transfected with BLTR1 siRNA. Although leukotriene B4 (LTB4) had minimal direct effects on MMD in control and 5-LO-deficient BMDCs, MMD attenuation by HMGB1 in 5-LO-deficient BMDCs was considerably reversed by exogenous LTB4, however, not in BLTR1-lacking BMDCs, recommending that LTB4/BLTR1-mediated priming of monocytes is normally a prerequisite of HMGB1-induced MMD. transfection Little interfering RNA (siRNA) for BLTR1 and scrambled siRNA (detrimental control) had been designed and synthesized utilizing a SilencerTM siRNA structure kit bought from Bioneer. The sequences of BLTR1 siRNA and scrambled siRNA had been 5-GAUCUGCGCUCCGAACUAUdTdT-3 and 5-AUAGUUCGGAGCGCAGAUCdTdT-3, respectively. For siRNA transfection, cells had been seeded and transfected with BLTR1 siRNA using Lipofectamine 2000 (Invitrogen, NY, USA) based on the manufacturer's process. Transfection efficiencies had been monitored utilizing a fluorescent oligonucleotide (BLOCK-iT Fluorescent Oligo; Invitrogen) and estimated to become between 80 and 90%. Immunofluorescence evaluation Wire-injured femoral arteries had been gathered and serial paraffin areas (4 m) of femoral arteries had been incubated with mouse-anti -SMA (1:400) and rabbit-anti Compact disc36 (1:200) antibodies. Alexa488-conjugated IgG and Alexa594-conjugated IgG (Abcam) had been utilized to detect immunofluorescence indicators for -SMA and Compact disc36, respectively. After nuclei had been visualized by staining with 0.1 g/ml diamidino-2-phenylindole (DAPI), slides had been mounted in Vectashield. Fluorescence pictures had been visualized by checking confocal microscopy (LSM 510, Carl Zeiss, Oberkochen, Germany), and analyzed by Country wide Institutes of Wellness (NIH) image software program (Picture J, NIH, USA). Transplantation of bone tissue marrow-derived monocytes BMDCs had been harvested in the femurs and tibiae of mice (7 wks, male), which have been euthanized by skin tightening and insufflation and cervical dislocation, and bone tissue marrow-derived monocytes (BMDMs, Compact disc11b-positive cells) had been after that separated using MACS technology (Miltenyi, Bergisch Gladbach, GER) utilizing a regular procedure. BMDCs had been after that stained with fluorochrome-labeled monoclonal anti-CD11b, sorted utilizing a BD ARIAIII cell sorter (Becton Dickinson, San Jose, CA, USA), cleaned, and resuspended at 1 107 cells/ml. Receiver BLTR1-lacking mice were implemented 1 107 BMDMs per mouse by tail vein shot. The expressions of BLTR1 mRNA and proteins in peripheral bloodstream monocytes (PBMCs) isolated from three sets of BMDMs-transplanted mice (WTWT mice, WT monocytes into WT mice; KOKO mice, BLTR1-lacking monocytes into BLTR1-lacking mice; and WTKO mice, WT monocytes into BLTR1-deficient mice) had been determined by REAL-TIME PCR and immunocytochemistry, respectively. Statistical evaluation Results were portrayed as means SEMs. One-way analysis of variance (ANOVA) accompanied by Turkey's multiple evaluation test was AS-605240 utilized to look for the significance of distinctions. Statistical significance was recognized for beliefs < 0.05. Outcomes A job for 5-LO in MMD induced by HMGB1 The consequences of HMGB1 over the appearance of 5-LO mRNA and proteins in BMDCs had been driven using semi-quantitative RT-PCR and Traditional western blot evaluation. In previous research, HMGB1 had been secreted to 10C100 ng/ml physiologically or pathologically (27, 28). Hence, BMDCs had been treated with HMGB1 at concentrations of 100 ng/ml inside our research. As proven in Figure ?Amount1A,1A, HMGB1 at focus of 100 ng/ml increased the mRNA and proteins appearance of 5-LO within a time-dependent way in BMDCs and THP-1 cells (Supplementary Amount 1), that have been attenuated by inhibition of varied receptors for HMGB1 (Supplementary Amount 2). To look for the useful function of 5-LO elevated in HMGB1-activated cells, LTB4 creation in HMGB1-treated cells was assessed using ELISA. As proven in Figure ?Amount1B,1B, LTB4 creation in HMGB1-stimulated cells was gradually increased up to 24 h (approximately 10 ng/107 cells), suggesting the participation of 5-LO-derived LTs in MMD induction by HMGB1. Open up in another window.vehicle. Open in another window Figure 3 Identification from the function played by BLTR1 in monocytes during HMGB1-induced MMD. by exogenous LTB4, however, not in BLTR1-deficient BMDCs, recommending that LTB4/BLTR1-mediated priming of monocytes is normally a prerequisite of HMGB1-induced MMD. transfection Little interfering RNA (siRNA) for BLTR1 and scrambled siRNA (detrimental control) had been designed and synthesized utilizing a SilencerTM siRNA structure kit bought from Bioneer. The sequences of BLTR1 siRNA and scrambled siRNA had been 5-GAUCUGCGCUCCGAACUAUdTdT-3 and 5-AUAGUUCGGAGCGCAGAUCdTdT-3, respectively. For siRNA transfection, cells had been seeded and transfected with BLTR1 siRNA using Lipofectamine 2000 (Invitrogen, NY, USA) based on the manufacturer's process. Transfection efficiencies had been monitored utilizing a fluorescent oligonucleotide (BLOCK-iT Fluorescent Oligo; Invitrogen) and estimated to become between 80 and 90%. Immunofluorescence evaluation Wire-injured femoral arteries had been gathered and serial paraffin areas (4 m) of femoral arteries had been incubated with mouse-anti -SMA (1:400) and rabbit-anti Compact disc36 (1:200) antibodies. Alexa488-conjugated IgG and Alexa594-conjugated IgG (Abcam) had been utilized to detect immunofluorescence indicators for -SMA and Compact disc36, respectively. After nuclei had been visualized by staining with 0.1 g/ml diamidino-2-phenylindole (DAPI), slides had been mounted in Vectashield. Fluorescence pictures had been visualized by checking confocal microscopy (LSM 510, Carl Zeiss, Oberkochen, Germany), and analyzed by Country wide Institutes of Wellness (NIH) image software program (Picture J, NIH, USA). Transplantation of bone tissue marrow-derived monocytes BMDCs had been harvested in the femurs and tibiae of mice (7 wks, male), which have been euthanized by skin tightening and insufflation and cervical dislocation, and bone tissue marrow-derived monocytes (BMDMs, Compact disc11b-positive cells) had been after that separated using MACS technology (Miltenyi, Bergisch Gladbach, GER) utilizing a regular procedure. BMDCs had been after that stained with fluorochrome-labeled monoclonal anti-CD11b, sorted utilizing a BD ARIAIII cell sorter (Becton Dickinson, San Jose, CA, USA), cleaned, and resuspended at 1 107 cells/ml. Receiver BLTR1-lacking mice were implemented 1 107 BMDMs per mouse by tail vein shot. The expressions of BLTR1 mRNA and proteins in peripheral blood monocytes (PBMCs) isolated from three groups of BMDMs-transplanted mice (WTWT mice, WT monocytes into WT mice; KOKO mice, BLTR1-deficient monocytes into BLTR1-deficient mice; and WTKO mice, WT monocytes into BLTR1-deficient mice) were determined by Real Time PCR and immunocytochemistry, respectively. Statistical analysis Results were expressed as means SEMs. One-way analysis of variance (ANOVA) followed by Turkey's multiple comparison test was used to determine the significance of differences. Statistical significance was accepted for values < 0.05. Results A role for 5-LO in MMD induced by HMGB1 The effects of HMGB1 around the expression of 5-LO mRNA and protein in BMDCs were decided using semi-quantitative RT-PCR and Western blot analysis. In previous studies, HMGB1 were secreted to 10C100 ng/ml physiologically or pathologically (27, 28). Thus, BMDCs were treated with HMGB1 at concentrations of 100 ng/ml in our study. As shown in Figure ?Physique1A,1A, HMGB1 at concentration of 100 ng/ml increased the mRNA and protein expression of 5-LO in a time-dependent manner in BMDCs and THP-1 cells (Supplementary Physique 1), which were attenuated by inhibition of various receptors for HMGB1 (Supplementary Physique 2). To determine the functional role of 5-LO increased in HMGB1-stimulated cells, LTB4 production in HMGB1-treated cells was measured using ELISA. As shown in Figure ?Physique1B,1B, LTB4 production in HMGB1-stimulated cells was gradually increased up to 24 h (approximately 10 ng/107 cells), suggesting the potential involvement of 5-LO-derived LTs in MMD induction by HMGB1. Open in a separate window Physique 1 Role of 5-LO in monocytes on monocyte-to-macrophage differentiation (MMD) induced by HMGB1. (A) Time-courses of the expressions of 5-LO mRNA and protein in BMDCs stimulated with HMGB1 (100 ng/ml) were decided using RT-PCR and Western blot, respectively. Bottom: Blot densities were quantified and presented as the means SEMs of 6C7 impartial experiments. *< 0.05; **< 0.01 vs. corresponding value at 0 h. (B) Time-course of LTB4 production in vehicle- or HMGB1 (100 ng/ml)-stimulated BMDCs as determined by ELISA. Bottom: Color signals were quantified, and data were presented as the means SEMs of 6C7 impartial experiments. *< 0.05; **< 0.01 vs. value at 0 h. (C) Representative immunocytochemical images of BMDCs stimulated with HMGB1. BMDCs were pre-treated with Zileuton at 10 or 30 M for 1 h, and then stimulated with HMGB1 (100 ng/ml) for 10 days. Cells were stained with anti-CD11b/Mac-1 (red) and.

BMDCs isolated from wild-type (WT) or BLTR1-deficient (KO) mice were stimulated with HMGB1 for 10 days, and then cellular expressions of CD11b/Mac-1 were determined by circulation cytometry