Journal: Journal of Inflammation Research

Article Title: P2X7 Receptor and Heart Function in a Mouse Model of Systemic Inflammation Due to High Fat Diet

doi: 10.2147/JIR.S356038

Figure Lengend Snippet: Heart gene expression profile of key regulators of oxidative stress modulation, endothelial function and chemotactic processes observed in wild-type mice treated with normal (WT-NFD) or high (WT-HFD) fat diet, or P2X7 receptor knockout mice treated with normal (KO-HFD) or high fat diet (KO-HFD). Tumor necrosis factor α (TNFα, A ), Nitric oxide synthase 2, inducible (NOS-2, B ), Monocyte chemotactic protein-1 (MCP-1, C ), Nitric oxide synthase 3, endothelial (NOS-3, D ), Adhesion G protein-coupled receptor E1 (ADGRE1, E ), Intercellular adhesion molecule 1 (ICAM-1, F ) are shown. Relative gene expression was calculated by the 2 −ΔΔCt method and normalized by the β-actin housekeeping gene. Results are reported as arbitrary units (A.U.). Data are presented as mean±SE for at least six animals in each group. Two-way ANOVA with genotype and diet as sources of variation, followed by Tukey’s post-hoc test, was used for multiple comparisons. Statistical significance was set at p < 0.05. ● WT-NFD, ■ WT-HFD, ▲ KO-NFD, ◆ KO-HFD.

Article Snippet: After blocking with BSA 5% in T-tris buffered saline (TBS and Tween-20 0.1%) for 1h at RT, blots were incubated overnight at 4°C with one of the following primary antibodies diluted 1:1000: anti-IL-1β (number 12242), anti-IL-6 (number 12912), and anti-p38 mitogen-activated protein kinase (p38MAPK) (number 9212), anti-p-p38MAPK (number 9211), anti-transforming growth factor-β (TGFβ) (number 3711), anti-monocyte chemoattractant protein-1 (MCP-1) (number 2029) from Cell Signaling Technology (Danvers, MA, USA); anti-NLRP3 (AG-20B-0014) from Adipogen (San Diego, CA, USA); anti-nitric oxide synthase-2 (NOS-2) (sc-651) from Santa Cruz Biotechnology (Dallas, TX, USA), and anti-tumor necrosis factor-α (TNFα) (PA5-19810) from ThermoFisher Scientific.

Techniques: Expressing, Knock-Out

Journal: Journal of Inflammation Research

Article Title: P2X7 Receptor and Heart Function in a Mouse Model of Systemic Inflammation Due to High Fat Diet

doi: 10.2147/JIR.S356038

Figure Lengend Snippet: Heart protein expression of key regulators of oxidative stress modulation, endothelial function and chemotactic processes whose genes were differentially expressed in wild-type mice treated with normal (WT-NFD) or high (WT-HFD) fat diet, or P2X7 receptor knockout mice treated with normal (KO-NFD) or high fat diet (KO-HFD). Tumor necrosis factor α (TNFα, A ), nitric oxide synthase 2, inducible (NOS-2, B ), Monocyte chemotactic protein-1 (MCP-1, C ) are shown. Band intensities were normalized based on reference protein GAPDH and results are expressed as arbitrary units (A.U.). Arrows indicate different MCP-1 isoforms. Data are presented as mean±SE for at least six animals in each group. Two-way ANOVA with genotype and diet as sources of variation, followed by Tukey’s post-hoc test, was used for multiple comparisons. Statistical significance was set at p < 0.05. ● WT-NFD, ■ WT-HFD, ▲ KO-NFD, ◆ KO-HFD.

Article Snippet: After blocking with BSA 5% in T-tris buffered saline (TBS and Tween-20 0.1%) for 1h at RT, blots were incubated overnight at 4°C with one of the following primary antibodies diluted 1:1000: anti-IL-1β (number 12242), anti-IL-6 (number 12912), and anti-p38 mitogen-activated protein kinase (p38MAPK) (number 9212), anti-p-p38MAPK (number 9211), anti-transforming growth factor-β (TGFβ) (number 3711), anti-monocyte chemoattractant protein-1 (MCP-1) (number 2029) from Cell Signaling Technology (Danvers, MA, USA); anti-NLRP3 (AG-20B-0014) from Adipogen (San Diego, CA, USA); anti-nitric oxide synthase-2 (NOS-2) (sc-651) from Santa Cruz Biotechnology (Dallas, TX, USA), and anti-tumor necrosis factor-α (TNFα) (PA5-19810) from ThermoFisher Scientific.

Techniques: Expressing, Knock-Out

Journal: Redox Biology

Article Title: Hydrogen sulfide as an anti-calcification stratagem in human aortic valve: Altered biogenesis and mitochondrial metabolism of H 2 S lead to H 2 S deficiency in calcific aortic valve disease

doi: 10.1016/j.redox.2023.102629

Figure Lengend Snippet: Decreased levels of bioavailable hydrogen sulfide and high expression of inflammatory cytokines are characteristics of human calcific aortic valves (A) Hematoxylin and eosin, TNF-α, IL-1β, and CSE immunohistochemical (IHC) stainings were performed on healthy aortic valves (HAVs) derived from the Department of Forensic Medicine, University of Debrecen (N = 5) and on calcific aortic valves (CAVs) of patients diagnosed with CAVD undergoing total aortic valve replacement (N = 5). Scale bars shown in the images represent 200 μm or 50 μm. (B) Quantitative analysis of TNF-α, and IL-1β IHC staining of tissue sections were calculated using ImageJ software (N = 5) (C, left panel). Quantitative analysis of CSE IHC staining of tissue sections was calculated using ImageJ software (N = 5) (C, right panel). Levels of bioavailable H 2 S of healthy aortic valves and calcific aortic valves measured with LC-MS/MS are shown (N = 5). Results were analyzed by unpaired t -test and are shown as mean values ± SEM of five independent experiments. ** p < 0.01; *** p < 0.001.

Article Snippet: For epitope retrieval, ULTRA Cell Conditioning 1 Solution (ULTRA CC1) was used (contains: Tris-based buffer and a preservative (Ventana medical systems; product no: 5424569001) at 100 °C, for 52 min. Antibodies used for IHC: rabbit anti-human TNF-α (Thermo Fisher Scientific; PA5-19810; 400 ng/mL); rabbit anti-human IL-1β (Invitrogen; 17 h18l16; 400 ng/mL); rabbit anti‐human CSE (Proteintech Group; 12217‐1‐AP; 1000 ng/mL); and rabbit anti-human CBS (Proteintech Group; 14787-1-AP; 700 μg/mL.

Techniques: Expressing, Immunohistochemical staining, Derivative Assay, Immunohistochemistry, Software, Liquid Chromatography with Mass Spectroscopy

Journal: Redox Biology

Article Title: Hydrogen sulfide as an anti-calcification stratagem in human aortic valve: Altered biogenesis and mitochondrial metabolism of H 2 S lead to H 2 S deficiency in calcific aortic valve disease

doi: 10.1016/j.redox.2023.102629

Figure Lengend Snippet: Mineralization potential and inflammatory response of valvular interstitial cells to high levels of phosphate Valvular interstitial cells (VICs) isolated from calcific aortic valves (CAV-VICs) or healthy aortic valves (HAV-VICs) were exposed to calcification medium supplemented with 2.5 mmol/L inorganic phosphate and 1.8 mmol/L calcium chloride every other day for 5 days. (A) Calcium contents of extracellular matrix normalized to a protein of VICs are shown (n = 3). (B) TNF-α levels normalized to GAPDH of VICs cultured in calcification medium or culture medium are shown (n = 3). (C) IL-1β levels normalized to GAPDH of VICs cultured in calcification medium or culture medium are shown (n = 3). Red lines indicate CAV-VICs cultured in calcification medium. Black lines indicate HAV-VICs cultured in calcification medium. Blue lines show the HAV-VICs maintained in culture medium. Results were analyzed by unpaired t -test and are shown as mean values ± SEM of three independent experiments. * p < 0.05; ** p < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: For epitope retrieval, ULTRA Cell Conditioning 1 Solution (ULTRA CC1) was used (contains: Tris-based buffer and a preservative (Ventana medical systems; product no: 5424569001) at 100 °C, for 52 min. Antibodies used for IHC: rabbit anti-human TNF-α (Thermo Fisher Scientific; PA5-19810; 400 ng/mL); rabbit anti-human IL-1β (Invitrogen; 17 h18l16; 400 ng/mL); rabbit anti‐human CSE (Proteintech Group; 12217‐1‐AP; 1000 ng/mL); and rabbit anti-human CBS (Proteintech Group; 14787-1-AP; 700 μg/mL.

Techniques: Isolation, Cell Culture

Journal: Redox Biology

Article Title: Hydrogen sulfide as an anti-calcification stratagem in human aortic valve: Altered biogenesis and mitochondrial metabolism of H 2 S lead to H 2 S deficiency in calcific aortic valve disease

doi: 10.1016/j.redox.2023.102629

Figure Lengend Snippet: Distinct calcification response of valvular interstitial cells to pro-inflammatory cytokines depending upon disease state Valvular interstitial cells (VICs) isolated from calcific aortic valves (CAV-VICs) or healthy aortic valves (HAV-VICs) were cultured in growth medium or calcification medium, containing 2.5 mmol/L inorganic phosphate and 1.8 mmol/L calcium chloride every other day, for 5 days. VICs maintained in calcification media were exposed to TNF-α (10 nmol/L) or IL-1β (10 nmol/L) for 5 days. (A) Alizarin Red S staining of CAV-VICs (upper panels) and HAV-VICs (lower panels) are shown. Quantitative analysis of Alizarin Red S staining of CAV-VICs and HAV-VICs was calculated using ImageJ software (n = 5). (B) Extracellular calcium contents normalized to protein of CAV-VICs and HAV-VICs are shown (n = 5). (C) ALP protein expressions normalized to GAPDH of CAV-VICs (left panels) and HAV-VICs (right panels) were measured with western blot (n = 3). Quantitative analysis of the ALP western blots were calculated using ImageJ software. (D) ALP staining of CAV-VICs (upper panels) and HAV-VICs (lower panels) are shown. Quantitative analysis of ALP staining of CAV-VICs and HAV-VICs was calculated using ImageJ software (n = 5). Results were analyzed by one-way ANOVA, Bonferroni's Multiple Comparison Test, and are shown as mean values ± SEM of three (D and E) or five (A–C) independent experiments. IL-1β and TNF-α treatment were compared to the minus condition (calcification medium). Ns: not significant; * p < 0.05; ** p < 0.01; *** p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: For epitope retrieval, ULTRA Cell Conditioning 1 Solution (ULTRA CC1) was used (contains: Tris-based buffer and a preservative (Ventana medical systems; product no: 5424569001) at 100 °C, for 52 min. Antibodies used for IHC: rabbit anti-human TNF-α (Thermo Fisher Scientific; PA5-19810; 400 ng/mL); rabbit anti-human IL-1β (Invitrogen; 17 h18l16; 400 ng/mL); rabbit anti‐human CSE (Proteintech Group; 12217‐1‐AP; 1000 ng/mL); and rabbit anti-human CBS (Proteintech Group; 14787-1-AP; 700 μg/mL.

Techniques: Isolation, Cell Culture, Staining, Software, Western Blot

Journal: Redox Biology

Article Title: Hydrogen sulfide as an anti-calcification stratagem in human aortic valve: Altered biogenesis and mitochondrial metabolism of H 2 S lead to H 2 S deficiency in calcific aortic valve disease

doi: 10.1016/j.redox.2023.102629

Figure Lengend Snippet: Transient inhibition of valvular interstitial cell calcification provoked by pro-inflammatory cytokines is associated with elevated cystathionine-γ-lyase expression and H 2 S generation. Valvular interstitial cells isolated from healthy aortic valves (HAV-VICs) were cultured in calcification medium, containing 2.5 mmol/L inorganic phosphate and 1.8 mmol/L calcium chloride every other day, for 5 and 14 days. VICs maintained in calcification media were exposed to TNF-α (10 nmol/L) or IL-1β (10 nmol/L) for 5 and 14 days. (A) Calcium contents of extracellular matrix normalized to protein of HAV-VICs are shown (n = 3). (B) CSE protein expression normalized to GAPDH in HAV-VICs maintained in calcification media with or without TNF-α (10 nmol/L) and IL-1β (10 nmol/L) for 5 and 14 days are shown (n = 3). (C) H 2 S generation measured with modified methylene blue method and normalized to protein of HAV-VICs are shown (n = 3). Results were analyzed by one-way ANOVA, Bonferroni's Multiple Comparison Test, and are shown as mean values ± SEM of three independent experiments. Ns: not significant; * p < 0.05; ** p < 0.01; *** p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: For epitope retrieval, ULTRA Cell Conditioning 1 Solution (ULTRA CC1) was used (contains: Tris-based buffer and a preservative (Ventana medical systems; product no: 5424569001) at 100 °C, for 52 min. Antibodies used for IHC: rabbit anti-human TNF-α (Thermo Fisher Scientific; PA5-19810; 400 ng/mL); rabbit anti-human IL-1β (Invitrogen; 17 h18l16; 400 ng/mL); rabbit anti‐human CSE (Proteintech Group; 12217‐1‐AP; 1000 ng/mL); and rabbit anti-human CBS (Proteintech Group; 14787-1-AP; 700 μg/mL.

Techniques: Inhibition, Expressing, Isolation, Cell Culture, Modification

Journal: Redox Biology

Article Title: Hydrogen sulfide as an anti-calcification stratagem in human aortic valve: Altered biogenesis and mitochondrial metabolism of H 2 S lead to H 2 S deficiency in calcific aortic valve disease

doi: 10.1016/j.redox.2023.102629

Figure Lengend Snippet: Cystathionine-γ-lyase derived hydrogen sulfide mediates the transient inhibition of calcification in healthy valvular interstitial cells triggered by pro-inflammatory cytokines CSE and CBS genes were silenced in valvular interstitial cells isolated from healthy aortic valves (HAV-VICs). After the silencing, cells were cultured in a growth medium or exposed to a calcification medium, containing 2.5 mmol/L inorganic phosphate and 1.8 mmol/L calcium chloride every other day, in the presence or absence of TNF-α (10 nmol/L) or IL-1β (10 nmol/L) for 5 days. (A) Alizarin Red S staining of HAV-VICs is shown. Quantitative analysis of Alizarin Red S staining of HAV-VICs was calculated using ImageJ software (n = 5). (A) Calcium contents of extracellular matrix normalized to protein of HAV-VICs are shown (n = 3). (B) CSE protein levels normalized to GAPDH in valvular interstitial cells isolated from calcific aortic valves (CAV-VICs) were measured with western blot (n = 3). Quantitative analysis of the CSE western blots was calculated using ImageJ software (n = 3). (C) H 2 S generation measured with modified methylene blue method and normalized to protein of CAV-VICs is shown (n = 5). Results were analyzed by one-way ANOVA, Bonferroni's Multiple Comparison Test, and are shown as mean values ± SEM. Ns: not significant; * p < 0.05; ** p < 0.01; *** p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: For epitope retrieval, ULTRA Cell Conditioning 1 Solution (ULTRA CC1) was used (contains: Tris-based buffer and a preservative (Ventana medical systems; product no: 5424569001) at 100 °C, for 52 min. Antibodies used for IHC: rabbit anti-human TNF-α (Thermo Fisher Scientific; PA5-19810; 400 ng/mL); rabbit anti-human IL-1β (Invitrogen; 17 h18l16; 400 ng/mL); rabbit anti‐human CSE (Proteintech Group; 12217‐1‐AP; 1000 ng/mL); and rabbit anti-human CBS (Proteintech Group; 14787-1-AP; 700 μg/mL.

Techniques: Derivative Assay, Inhibition, Isolation, Cell Culture, Staining, Software, Western Blot, Modification

Journal: Redox Biology

Article Title: Hydrogen sulfide as an anti-calcification stratagem in human aortic valve: Altered biogenesis and mitochondrial metabolism of H 2 S lead to H 2 S deficiency in calcific aortic valve disease

doi: 10.1016/j.redox.2023.102629

Figure Lengend Snippet: Mitochondria-targeting H 2 S donor inhibits inflammation of valvular interstitial cells Valvular interstitial cells (VICs) isolated from healthy aortic valves (HAV-VICs) were cultured in growth medium or calcification medium, containing 2.5 mmol/L inorganic phosphate and 1.8 mmol/L calcium chloride every other day, in the presence or absence of [10-oxo-10-[4-(3-thioxo-3H-1,2-dithiol-5-yl)phenoxy]decyl]triphenyl-phosphonium (AP39) (5 nmol/L) for 5 days. (A) Relative expression of IL-1β (left panel) and TNF-α (right panel) were analyzed by Real-Time qPCR (n = 3) in HAV-VICs are shown. (B) Protein expression of IL-1β, TNF-α, and GAPDH were determined by western blots in HAV-VICs are shown. Quantification of IL-1β and TNF-α optical density was calculated by ImageJ software. (C) HAV-VICs grown on coverslips and cultured in growth medium or calcification medium, containing 2.5 mmol/L inorganic phosphate and 1.8 mmol/L calcium chloride every other day, in the presence or absence of AP39 (5 nmol/L) for 5 days. Cells were stained with Hoechst 33258 for DNA (blue), an anti-IL-1β antibody with Alexa Fluor 488 secondary antibody for IL-1β (green), an anti-TNF-α antibody with Alexa Fluor 488 secondary antibody for TNF-α (green), and with Osteosense Fluorescent Probe (red). Images were taken with Leica TCS SP8 gated STED-CW nanoscopy. Images were deconvolved using Huygens Professional software. The color intensity of IL-1β, TNF-α, and Osteosense stainings was calculated using ImageJ software. Representative image, n = 5. Scale bars shown in the images represent 50 μm. Results were analyzed by one-way ANOVA, Bonferroni's Multiple Comparison Test, and were shown as mean values ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: For epitope retrieval, ULTRA Cell Conditioning 1 Solution (ULTRA CC1) was used (contains: Tris-based buffer and a preservative (Ventana medical systems; product no: 5424569001) at 100 °C, for 52 min. Antibodies used for IHC: rabbit anti-human TNF-α (Thermo Fisher Scientific; PA5-19810; 400 ng/mL); rabbit anti-human IL-1β (Invitrogen; 17 h18l16; 400 ng/mL); rabbit anti‐human CSE (Proteintech Group; 12217‐1‐AP; 1000 ng/mL); and rabbit anti-human CBS (Proteintech Group; 14787-1-AP; 700 μg/mL.

Techniques: Isolation, Cell Culture, Expressing, Western Blot, Software, Staining

Journal: Antioxidants & Redox Signaling

Article Title: Oxidation of Hemoglobin Drives a Proatherogenic Polarization of Macrophages in Human Atherosclerosis

doi: 10.1089/ars.2020.8234

Figure Lengend Snippet: Cellular responses in human complicated plaque of the carotid artery. (A, B) The area of a fresh hemorrhagic region (A, B, left column ) was compared with a hemorrhagic transformed region (A, B, right column ) using immunohistochemistry. Glycophorin A positivity demonstrates intact red blood cells (fresh hemorrhage) within a lesion, whereas Prussian blue indicates iron accumulation (hemosiderin) in the hemorrhagic transformed area. The presence and activation of macrophages are indicated by markers of CD68, carboxypeptidase M/CPM, TNF-α, and IL-1β (at 100 × magnification). (C) CD68 and glycophorin A costaining was performed on complicated lesions. Images of fresh hemorrhagic area ( left image ) and hemorrhagic transformed region ( right image ) were shown. The mean intensity of CD68 and glycophorin A stainings was calculated using ImageJ software ( N = 5). Heme-responsive proteins such as HO-1 and H-ferritin are demonstrated. (C–E) Quantitative analysis of immunohistochemical stainings of tissue sections was performed using ImageJ software ( N = 5). Scale bars shown in the images represent 50 μm. ** p < 0.01; *** p < 0.001. HO-1, heme oxygenase-1. Color images are available online.

Article Snippet: Kft, KP-1 clone) without dilution (ready to use) for 1 h; with the glycophorin A antibody (M081901-2, clone: JC159; Dako, an Agilent Technologies Company) at a dilution of 1:1000 for 1 h; with the carboxypeptidase M antibody (ab150405 clone: EPR8052; Abcam Plc, Cambridge, United Kingdom) at a dilution of 1:200 for 1 h; with the HMOX1 antibody (10701-1-AP, polyclonal; Invitrogen) at a dilution of 1:400 for 1 h; with the ferritin heavy chain antibody (ab75972 clone: EPR3005Y; Abcam Plc) at a dilution of 1:50 for 1 h; with the IL-1 beta antibody (P420B, polyclonal ab; Invitrogen) at a dilution of 1:100 for 1 h; with the TNF-α antibody (PA5-19810, polyclonal ab; Invitrogen) at a dilution of 1:100 for 1 h, and with the mouse anti-human ferrylHb antibody (made by our groups) at a dilution of 1:100 for 1 h. Staining was performed in a Ventana BenchMark Ultra immunostainer (Roche Diagnostics) in the following circumstances: Antibody Diluent, Cat. No. 251-018 (contains 0.3% protein in 0.1 M phosphate-buffered saline [pH 7.3] and 0.05% ProClin 300, a preservative).

Techniques: Transformation Assay, Immunohistochemistry, Activation Assay, Software, Immunohistochemical staining

Journal: Antioxidants & Redox Signaling

Article Title: Oxidation of Hemoglobin Drives a Proatherogenic Polarization of Macrophages in Human Atherosclerosis

doi: 10.1089/ars.2020.8234

Figure Lengend Snippet: Ferryl hemoglobin-positive macrophages in carotid artery are positive for IL-1β and TNF-α. Cross sections of a healthy carotid artery and atheromatous and hemorrhagic transformed lesions were shown. (A) Images demonstrated macrophages positive for both CD68 and IL-1β in the atheromatous plaque and positive for ferrylHb, CD68, and IL-1β in the hemorrhagic transformed lesions. Sections were stained with Hoechst 33258 for DNA ( blue ), an anti-ferrylHb antibody with Alexa Flour 488 secondary antibody for ferrylHb ( green ), an anti-CD68 antibody with Alexa Flour 568 secondary antibody for CD68 ( yellow ), and an anti-IL-1β antibody with Alexa Flour 647 secondary antibody for IL-1β ( red ). (B) Images demonstrated macrophages positive for both CD68 and TNF-α in the atheromatous plaque and positive for ferrylHb, CD68, and TNF-α in the hemorrhagic transformed lesions. Sections were stained with Hoechst 33258 for DNA ( blue ), an anti-ferrylHb antibody with Alexa Flour 488 secondary antibody for ferrylHb ( green ), an anti-CD68 antibody with Alexa Flour 568 secondary antibody for CD68 ( yellow ), and an anti-TNF-α antibody with Alexa Flour 647 secondary antibody for TNF-α ( red ). Images were taken using Leica TCS SP8 gated STED-CW nanoscopy. Images were deconvolved using Huygens Professional software. Representative image, N = 5. (C) Fluorescence intensity for ferrylHb ( n = 17), CD68 ( n = 20), IL-1β ( n = 19), and TNF-α ( n = 15) staining was calculated using ImageJ software. * p < 0.05; ** p < 0.01; *** p < 0.001. Color images are available online.

Article Snippet: Kft, KP-1 clone) without dilution (ready to use) for 1 h; with the glycophorin A antibody (M081901-2, clone: JC159; Dako, an Agilent Technologies Company) at a dilution of 1:1000 for 1 h; with the carboxypeptidase M antibody (ab150405 clone: EPR8052; Abcam Plc, Cambridge, United Kingdom) at a dilution of 1:200 for 1 h; with the HMOX1 antibody (10701-1-AP, polyclonal; Invitrogen) at a dilution of 1:400 for 1 h; with the ferritin heavy chain antibody (ab75972 clone: EPR3005Y; Abcam Plc) at a dilution of 1:50 for 1 h; with the IL-1 beta antibody (P420B, polyclonal ab; Invitrogen) at a dilution of 1:100 for 1 h; with the TNF-α antibody (PA5-19810, polyclonal ab; Invitrogen) at a dilution of 1:100 for 1 h, and with the mouse anti-human ferrylHb antibody (made by our groups) at a dilution of 1:100 for 1 h. Staining was performed in a Ventana BenchMark Ultra immunostainer (Roche Diagnostics) in the following circumstances: Antibody Diluent, Cat. No. 251-018 (contains 0.3% protein in 0.1 M phosphate-buffered saline [pH 7.3] and 0.05% ProClin 300, a preservative).

Techniques: Transformation Assay, Staining, Software, Fluorescence

Journal: Antioxidants & Redox Signaling

Article Title: Oxidation of Hemoglobin Drives a Proatherogenic Polarization of Macrophages in Human Atherosclerosis

doi: 10.1089/ars.2020.8234

Figure Lengend Snippet: Transcriptomic analysis reveals that ferryl hemoglobin polarizes macrophages into a proinflammatory-like phenotype. (A, B) RNA-seq transcriptomic analysis was performed on human macrophages treated with ferrylHb (10 μ M ) for 8 h ( n = 4/group). (A) Heatmap of the 50 most-changed DE genes is shown in log 10 (FC). (B) M1 and M2 signatures are shown as a clustered heatmap in log 10 (FC). (C, D) Relative expression of (C) IL-1β, TNF-α, CXCL8, (D) CD209, and IL-27RA were analyzed by real-time qPCR ( n = 3). (E) Protein expression of HO-1, H-ferritin, Pro-IL-1β, IL-1β, TNF-α and GAPDH was determined by Western blots in macrophages exposed to ferrylHb (10 μ M , 8-h ferrylHb treatment). ** p < 0.01; *** p < 0.001. DE, differentially expressed; FC, fold change. Color images are available online.

Article Snippet: Kft, KP-1 clone) without dilution (ready to use) for 1 h; with the glycophorin A antibody (M081901-2, clone: JC159; Dako, an Agilent Technologies Company) at a dilution of 1:1000 for 1 h; with the carboxypeptidase M antibody (ab150405 clone: EPR8052; Abcam Plc, Cambridge, United Kingdom) at a dilution of 1:200 for 1 h; with the HMOX1 antibody (10701-1-AP, polyclonal; Invitrogen) at a dilution of 1:400 for 1 h; with the ferritin heavy chain antibody (ab75972 clone: EPR3005Y; Abcam Plc) at a dilution of 1:50 for 1 h; with the IL-1 beta antibody (P420B, polyclonal ab; Invitrogen) at a dilution of 1:100 for 1 h; with the TNF-α antibody (PA5-19810, polyclonal ab; Invitrogen) at a dilution of 1:100 for 1 h, and with the mouse anti-human ferrylHb antibody (made by our groups) at a dilution of 1:100 for 1 h. Staining was performed in a Ventana BenchMark Ultra immunostainer (Roche Diagnostics) in the following circumstances: Antibody Diluent, Cat. No. 251-018 (contains 0.3% protein in 0.1 M phosphate-buffered saline [pH 7.3] and 0.05% ProClin 300, a preservative).

Techniques: RNA Sequencing Assay, Expressing, Western Blot