Journal: International journal of medical sciences

Article Title: Suppressive effects of S100A8 and S100A9 on neutrophil apoptosis by cytokine release of human bronchial epithelial cells in asthma.

doi: 10.7150/ijms.37833

Figure Lengend Snippet: Figure 2. S100A8 and S100A9 induce the secretion of MCP-1, IL-6 and IL-8 through TLR4, PI3K, AKT, MAPKs and NF-κB in BEAS-2B cells. (A-B) BEAS-2B cells were pre-treated for 1 h with or without 5 μM TLR4i, 5 μM LY294002 (LY), 10 μM AKTi (AKT), 10 μM PD98059 (PD), 10 μM SB202190 (SB), 10 μM SP600125 (SP) and 2 μM BAY-11-7085 (BAY), after which the cells were incubated for 48 h in the absence or presence of 10 µg/mL S100A8 (A) and S100A9 (B). The supernatant (n=4) was collected and analyzed by ELISA. Data are expressed as the means ± SD and are presented relative to the control set at 100%. *p < 0.05 and **p < 0.01 indicate a significant difference between the control and S100A8- or S100A9-treated groups, and ##p < 0.01 and #p < 0.05 represent a significant difference between the S100A8- or S100A9-treated groups and the inhibitor-treated groups.

Article Snippet: TLR4 inhibitor (TLR4i), CLI-095, was purchased from Invivogen (San Diego, CA, USA).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Control

Journal: International journal of medical sciences

Article Title: Suppressive effects of S100A8 and S100A9 on neutrophil apoptosis by cytokine release of human bronchial epithelial cells in asthma.

doi: 10.7150/ijms.37833

Figure Lengend Snippet: Figure 4. Expression of TLR4 in BEAS-2B cells after treatment with S100A8 and S100A9. BEAS-2B cells (n=3) were treated with 10 μg/mL S100A8 (A) and S100A9 (B) for the indicated time, and the expression of TLR4 was analyzed by flow cytometry. Data are expressed as the means ± SD, and are presented relative to the control set at 1. *p < 0.05 and **p < 0.01 indicate a significant difference.

Article Snippet: TLR4 inhibitor (TLR4i), CLI-095, was purchased from Invivogen (San Diego, CA, USA).

Techniques: Expressing, Flow Cytometry, Control

Journal: Nature Communications

Article Title: Lung-derived HMGB1 is detrimental for vascular remodeling of metabolically imbalanced arterial macrophages

doi: 10.1038/s41467-020-18088-2

Figure Lengend Snippet: Oxygen consumption rate (OCR) and respiration quantifications of WT and Ripk3 −/− BMDM treated with HMGB1 ( a , b ). n = 3 (WT + HMGB1, Ripk3 −/− and Ripk3 −/− +HMGB1) and 7 (WT). * P < 0.05, *** P < 0.001. OCR profiles and quantification of WT and Ripk3 −/− BMDM treated with HMGB1 in the presence of GSK’872 (10 µM, 1 h) ( c , d ). n = 5 per group. ** P < 0.01, *** P < 0.001. Representative histograms of Mitotracker and quantification of the mean fluorescence intensity (MFI) of WT or Ripk3 −/− BMDM treated with HMGB1 ( e ). n = 3 ( Ripk3 −/− and Ripk3 −/− +HMGB1), 5 (WT + HMGB1) and 6 (WT). * P < 0.05, *** P < 0.001. Representative histograms of Mitotracker and quantification of MFI of WT BMDM treated with lung extracts of PBS-treated mice (CTL) or ALI lung extracts (ALI) pre-incubated with anti-HMGB1 (α-HMGB1) or with control IgG ( f ). n = 3 per group. * P < 0.05. ( g ) Representative electron microscopy images of mitochondria in WT or Ripk3 −/− BMDM treated with HMGB1 or PBS (CTL). Scale bar = 500 nm. Quantification of mitochondria number per cell ( h ) in WT or Ripk3 −/− as indicated. n = 11 (WT), 10 (WT + HMGB1), 12 ( Ripk3 −/− ) and (17 Ripk3 −/− +HMGB1). ** P < 0.01. ( i ) IF staining of Mitotracker and quantification of mean mitochondrial area in WT or Ripk3 −/− BMDM treated with HMGB1 (10 ng ml −1 ). Scale bar = 10 µm. n = 4 per group. * P = 0.0358. ( j ) Immunoblot of pDRP1 and quantification. n = 3 per group . * P < 0.05. ( k ) Representative IF of CD68 (green) and pDRP1 (red) staining in aortas of mice as indicated (scale bar = 20 µm). Inset show magnified images (scale bar = 10 µm) ( i ) Schematic representation of Mdivi-1 treatment in mice. i.n., intranasal; i.p., intraperitoneal. Aneurysm incidence ( m ), quantification of maximal aortic diameter ( n ), representative color Doppler ultrasound images of aorta and M-mode (arrow) ( o ) and Verhoeff-Van Gieson staining ( p ) of aortic sections from vehicle or Mdivi-1 treatment. Arrows indicates elastin breaks. n = 4 (Mdivi-1) and 6 (Vehicle). *** P = 0.0002. Data is presented as mean, error bars represent s.e.m. P values were calculated using two-tailed unpaired t -tests ( i , n ) or one-way ANOVA ( b – h , j ).

Article Snippet: BMDM were pre-treated with pan-caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone (Z-VAD FMK, Bachem N-1510, 20 µM), DRP1 inhibitor Mdivi-1 (BML-CM127-0050, Enzolife Science, 10 µM), RIPK3 inhibitor GSK’872 (6492, R&D Systems), mitochondrial ROS scavenger MitoTEMPO (16621, Cayman Chemical, 10 µM) or TLR4 inhibitor (TLR4i) CLI-095 (tlrl-cli95, InvivoGen, 1 µg ml −1 ) in assays as indicated.

Techniques: Fluorescence, Incubation, Electron Microscopy, Staining, Western Blot, Two Tailed Test

Journal: Nature Communications

Article Title: Lung-derived HMGB1 is detrimental for vascular remodeling of metabolically imbalanced arterial macrophages

doi: 10.1038/s41467-020-18088-2

Figure Lengend Snippet: MitoSOX quantification of WT and Ripk3 −/− BMDM treated with HMGB1 ( a ) and WT BMDM treated with HMGB1 in the presence or not of Mdivi-1 or MitoTEMPO ( b ). n = 3 . * P < 0.05, *** P < 0.001. ( c ) Relative mRNA expression of Glud1 in WT or Ripk3 −/− BMDM. n = 3 per group . * P < 0.05, *** P < 0.001. ( d ) Representative IF images of dihydroethidium (DHE) staining (red) of aortic sections of mice treated as indicated. Elastin autofluorescence (green, scale bar = 20 µm). ( e ) Heatmap representation of differentially expressed genes in macrophages expressing Ripk3 ( Ripk3 + ) or not ( Ripk3 − ). ( f ) qPCR analysis of Mmp12 mRNA in WT or Ripk3 −/− aortas. n = 3 per group. ** P < 0.01, *** P < 0.001. ( g ) Spider graph representation (Log 10 scale) of multiplex analysis of MMPs in aortic extracts from WT or Ripk3 −/− mice. n = 3 ( Ripk3 −/− ) and 4 (WT). * P = 0.0454. Representative image of IF staining of MMP12, and its colocalization with CD68 macrophages in aortic section of mice treated as indicated ( h , i ). Scale bar = 20 µm. ( j ) Mmp12 mRNA in WT or Ripk3 −/− BMDM treated with HMGB1 (10 ng ml −1 ) in the presence or not of Mdivi-1 (10 µM) or MitoTEMPO (10 µM). n = 3 per group. * P < 0.05, ** P < 0.01. ( k ) Schematic representation of experimental protocol (top) and incidence of AAA in WT and Mmp12 −/− mice (below). i.n. intranasal. n = 4–5 per group. Representative color Doppler ultrasound images of aorta and M-mode screenshots (arrow) ( i ). Chronological quantifications of aortic diameter ( m ), maximal aortic diameter ( n ), and representative Verhoeff-Van Gieson staining in aortic sections of WT and Mmp12 −/− mice in the presence of ALI ( o ). n = 3 per group. Arrows indicate elastin breaks. Data is presented as mean, error bars represent s.e.m. (scale bar = 50 µm). * P = 0.0364. P va/clues were calculated by one-tailed ( g ) or two-tailed ( m , n ) unpaired t -tests, or one-way ANOVA ( a – f , j ).

Article Snippet: BMDM were pre-treated with pan-caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone (Z-VAD FMK, Bachem N-1510, 20 µM), DRP1 inhibitor Mdivi-1 (BML-CM127-0050, Enzolife Science, 10 µM), RIPK3 inhibitor GSK’872 (6492, R&D Systems), mitochondrial ROS scavenger MitoTEMPO (16621, Cayman Chemical, 10 µM) or TLR4 inhibitor (TLR4i) CLI-095 (tlrl-cli95, InvivoGen, 1 µg ml −1 ) in assays as indicated.

Techniques: Expressing, Staining, Multiplex Assay, One-tailed Test, Two Tailed Test

Journal: Nature Communications

Article Title: Lung-derived HMGB1 is detrimental for vascular remodeling of metabolically imbalanced arterial macrophages

doi: 10.1038/s41467-020-18088-2

Figure Lengend Snippet: HMGB1 derived from injured lungs is captured by TLR4 transmural macrophages in the abdominal aorta. Activation of TLR4 by HMGB1 upregulates RIPK3 and activates its phosphorylation. RIPK3 activates phospho-DRP1 which triggers mitochondrial fission and increases mitochondrial reactive oxygen species (ROS) production. ROS stimulates the expression of MMP12 by macrophages responsible for elastin fiber degradation in the aorta. Inhibition of phosphorylation of RIPK3 on Serine 204 refrains DRP1 activation and subsequent MMP12 expression.

Article Snippet: BMDM were pre-treated with pan-caspase inhibitor carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone (Z-VAD FMK, Bachem N-1510, 20 µM), DRP1 inhibitor Mdivi-1 (BML-CM127-0050, Enzolife Science, 10 µM), RIPK3 inhibitor GSK’872 (6492, R&D Systems), mitochondrial ROS scavenger MitoTEMPO (16621, Cayman Chemical, 10 µM) or TLR4 inhibitor (TLR4i) CLI-095 (tlrl-cli95, InvivoGen, 1 µg ml −1 ) in assays as indicated.

Techniques: Derivative Assay, Activation Assay, Expressing, Inhibition

Journal: Molecular medicine reports

Article Title: Effect of S100A8 and S100A9 on expressions of cytokine and skin barrier protein in human keratinocytes.

doi: 10.3892/mmr.2019.10454

Figure Lengend Snippet: Figure 6. HaCaT cells were treated with (A) S100A8 or (B) S100A9 (10 µg/ml) for 24 h and TLR4 expression was increased as assessed by western blotting. Data are expressed as the mean ± standard deviation. **P<0.01 vs. the control. S100, calcium binding protein; TLR4, toll‑like receptor 4.

Article Snippet: Tlr4 inhibitor cli-095 (Tlr4i), protein kinase δ (PKcδ) inhibitor (rottlerin), p38 mitogen-activated Correspondence to: dr in Sik Kim, department of Biomedical laboratory Science, School of Medicine, eulji university, 77 Gyeryoung-ro 771 beon-gil, Jung-Gu, daejeon 34824, republic of Korea e-mail: orientree@eulji.ac.kr Key words: atopic dermatitis, S100 calcium binding protein a8, S100 calcium binding protein a9, keratinocyte, skin barrier protein protein kinase (MaPK) inhibitor (SB202190), MeK inhibitor (Pd98059) and nuclear factor-κB (nF-κB) inhibitor (BaY-11-7085) were obtained from calbiochem (Merck KGaa). antibodies against p38 MaPK (cat. no. 9212), phospho-p38 MaPK (cat. no. 9211), phospho-extracellular signal regulated kinase 1/2 (erK1/2; cat. no. 9101), rabbit igG-HrP (cat. no. 7074), and mouse igG-HrP (cat. no. 7076) were acquired from cell Signaling Technology, inc. anti-erK2 (cat. no. sc-154) and anti-Tlr4 (cat. no. sc-10741) antibodies were obtained from Santa cruz Biotechnology, inc. Production of recombinant S100A8 and S100A9 proteins. in our previous report, the cdna of human S100a8 and S100a9 was cloned into peT28 expression vector (Merck KGaa) (10). recombinant S100a8 and S100a9 expression was induced with 1 mM isopropyl β-d-thiogalactoside in E. coli Bl21 (de3; Merck KGaa).

Techniques: Expressing, Western Blot, Standard Deviation, Control, Binding Assay