Journal: Redox Biology

Article Title: Nogo-B mediates endothelial oxidative stress and inflammation to promote coronary atherosclerosis in pressure-overloaded mouse hearts

doi: 10.1016/j.redox.2023.102944

Figure Lengend Snippet: Nogo-B mediated excess ROS production in HUVECs upon TNF-α stimulation. ( A ) Representative confocal images of MitoSOX Red fluorescence in HUVECs. (B ) Flow cytometric analysis of MitoSOX Red staining-indicated mitochondrial ROS level and quantification by MFI from scrambled (NC) or Nogo-B siRNA under vehicle or TNFα stimuli for 24 h n = 8/group. ( C ) Representative images of DCF fluorescence and ( D ) Intensity statistics and distribution of DCF fluorescence images in HUVECs under 24-h TNFα stimulation. n ≥ 80 (DCF-DA Green) cells from 6 independent experiments. ( E ) representative traces and ( F ) statistics of HPLC analysis of DHE oxidative products in HUVECs lysates transfected with scrambled (NC) or Nogo-B siRNA under vehicle or TNFα stimuli for 24 h. n = 7 independent experiments for each group. ( G ) NOX activity assays in TNFα-stimulated HUVECs transfected with NC or si-Nogo-B. n = 5/group. ( H ) Western blot analysis of NOX2 in TNFα-stimulated HUVECs transfected with NC or si-Nogo-B. n = 5/group. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Polyvinylidene fluoride membranes were incubated at 4 °C overnight using the following antibodies: Nogo-A/B (1:2000, #ab47085, Abcam); VCAM-1 (1:2000, #T55427, Abmart); ICAM-1 (1:500, #T40024, Abmart); P38 (1:1000, #9212, CST); p-P38 (1:1000, #9216, CST); P65 (1:1000, #8242, CST); p-P65 (1:1000, #3033, CST); AKT (1:1000, #9272, CST); P-AKT (1:1000, #9271, CST); IP3R1 (1:500, #ACC-019, Alomone); NgBR (1:1000, #TN25175S, Abmart); NOX2 (1:1000, #19013-1-AP, proteintech); NOX1 (1:1000, #17772-1-AP, Proteintech); NOX4 (1:1000, #14347-1-AP, proteintech); OPA1 (1:1000, #67589, CST); Drp1 (1:1000, #5391, CST); Mfn1 (1:1000, #66776-1-Ig, proteintech); Mfn2 (1:1000, #12186-1-AP, proteintech); VDAC1 (1:2000, #66345-1-Ig, proteintech); MCU(1:2000, #26312-1-AP, proteintech); Fis1(1:500, #10956-1-AP, proteintech) and GAPDH (1:5000, #HRP-60004, proteintech), followed by the rabbit or mouse with HRP-conjugated secondary antibody.

Techniques: Fluorescence, Staining, Transfection, Activity Assay, Western Blot

Journal: Redox Biology

Article Title: Nogo-B mediates endothelial oxidative stress and inflammation to promote coronary atherosclerosis in pressure-overloaded mouse hearts

doi: 10.1016/j.redox.2023.102944

Figure Lengend Snippet: Nogo-B-activated ROS-p38-p65 pathway was responsible for adhesion molecules upregulation in ECs. ( A-B ) Reprsentative Western blotting images and quantifications of protein levels of VCAM-1 and Nogo-B in HUVECs transfected with NC or si-Nogo-B upon 24-h TNFα stimulation in the presence or absence of 20 μM Mitotempo ( A, n = 7/group) or 5 mM NAC ( B, n = 5/group). ( C ) Western blot analysis of VCAM-1, NOX2 and Nogo-B in HUVECs under 24-h TNFα stimuli in the presence or absence of 50 μM GSK2795039 (NOX2 inhibitors). n = 7 per group. ( D-E ) Western blot analysis of VCAM-1 and Nogo-B in whole heart tissue after 24-h TAC treated with vehicle or NAC or infliximab (a chimeric anti-TNFα monoclonal antibody). n = 5/group.

Article Snippet: Polyvinylidene fluoride membranes were incubated at 4 °C overnight using the following antibodies: Nogo-A/B (1:2000, #ab47085, Abcam); VCAM-1 (1:2000, #T55427, Abmart); ICAM-1 (1:500, #T40024, Abmart); P38 (1:1000, #9212, CST); p-P38 (1:1000, #9216, CST); P65 (1:1000, #8242, CST); p-P65 (1:1000, #3033, CST); AKT (1:1000, #9272, CST); P-AKT (1:1000, #9271, CST); IP3R1 (1:500, #ACC-019, Alomone); NgBR (1:1000, #TN25175S, Abmart); NOX2 (1:1000, #19013-1-AP, proteintech); NOX1 (1:1000, #17772-1-AP, Proteintech); NOX4 (1:1000, #14347-1-AP, proteintech); OPA1 (1:1000, #67589, CST); Drp1 (1:1000, #5391, CST); Mfn1 (1:1000, #66776-1-Ig, proteintech); Mfn2 (1:1000, #12186-1-AP, proteintech); VDAC1 (1:2000, #66345-1-Ig, proteintech); MCU(1:2000, #26312-1-AP, proteintech); Fis1(1:500, #10956-1-AP, proteintech) and GAPDH (1:5000, #HRP-60004, proteintech), followed by the rabbit or mouse with HRP-conjugated secondary antibody.

Techniques: Western Blot, Transfection

Journal: Frontiers in Immunology

Article Title: Identification of biomarkers related to immune and inflammation in membranous nephropathy: comprehensive bioinformatic analysis and validation

doi: 10.3389/fimmu.2023.1252347

Figure Lengend Snippet: Analysis of immune-associated hub genes. (A) Lollipop plot of CSF1R correlation with 28 immune cells. The horizontal coordinate indicates the correlation size, and the color indicates the P-value. (B) Immune-related GSEA analysis of CSF1R with different expression levels. (C) Distribution of 28 immune cells in CSF1R with different expression levels. (D-F) Analysis of CYBB. **P <0.01, ***P <0.001, ****P <0.0001.

Article Snippet: To measure the protein levels of CSF-1 and CYBB/NOX2, we used specific ELISA kits for human CSF-1 (KE00184; Proteintech, USA) and CYBB/NOX2 (EK13559; Signalway Antibody, USA).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Identification of biomarkers related to immune and inflammation in membranous nephropathy: comprehensive bioinformatic analysis and validation

doi: 10.3389/fimmu.2023.1252347

Figure Lengend Snippet: Validation of CSF-1 and CYBB. Each point represents a sample. (A, B) ELISA measurement of CSF-1 and CYBB. (C, D) mRNA expression of CSF1R and CYBB was analyzed by RT-qPCR. *** P < 0.001, vs Control.

Article Snippet: To measure the protein levels of CSF-1 and CYBB/NOX2, we used specific ELISA kits for human CSF-1 (KE00184; Proteintech, USA) and CYBB/NOX2 (EK13559; Signalway Antibody, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR

Journal: Apoptosis

Article Title: Glucocorticoid-induced activation of NOX/ROS/NF-κB signaling in MSCs contributes to the development of GONFH

doi: 10.1007/s10495-023-01860-2

Figure Lengend Snippet: Primer sequences for RT-qPCR.

Article Snippet: Subsequently, sections were incubated with anti-RUNX2 (dilution 1:200, AB236639; Abcam, Cambridge, UK), anti-OSX (dilution 1:200, ER1914-47; Huabio, Hangzhou, Zhejiang, China), anti-PPARγ (dilution 1:300, AB19481; Abcam, Cambridge, UK), anti-fatty acid binding protein-4 (FABP4, dilution 1:300, AB92501; Abcam, Cambridge, UK), anti-NOX2 (dilution 1:500, 19013-1-AP; Proteintech, Chicago, USA), anti-NOX4 (dilution 1.

Techniques: Sequencing

Journal: Apoptosis

Article Title: Glucocorticoid-induced activation of NOX/ROS/NF-κB signaling in MSCs contributes to the development of GONFH

doi: 10.1007/s10495-023-01860-2

Figure Lengend Snippet: GCs-induced exacerbation of the OS microenvironment in the necrotic region of GONFH, with enhanced NOX protein expressions and ROS levels. ( A ) ROS staining and quantitative analysis of human femoral head. ( B ) ROS staining and quantitative analysis of rat femoral head. ( C ) ROS staining and quantitative analysis of MSCs treated with 1µM DEX. ( D ) Immunohistochemistry staining and quantitative analysis of NOX2 and NOX4 of human femoral head. ( E ) Immunohistochemistry staining and quantitative analysis of NOX2 and NOX4 of rat femoral head. ( F ) NOX2 and NOX4 mRNA of MSCs treated with 1µM DEX. ( G ) NOX2 and NOX4 protein levels of MSCs treated with 1µM DEX. PC, positive control, *** P < 0.001 (vs. GONFH or DEX).

Article Snippet: Subsequently, sections were incubated with anti-RUNX2 (dilution 1:200, AB236639; Abcam, Cambridge, UK), anti-OSX (dilution 1:200, ER1914-47; Huabio, Hangzhou, Zhejiang, China), anti-PPARγ (dilution 1:300, AB19481; Abcam, Cambridge, UK), anti-fatty acid binding protein-4 (FABP4, dilution 1:300, AB92501; Abcam, Cambridge, UK), anti-NOX2 (dilution 1:500, 19013-1-AP; Proteintech, Chicago, USA), anti-NOX4 (dilution 1.

Techniques: Staining, Immunohistochemistry, Positive Control

Journal: Apoptosis

Article Title: Glucocorticoid-induced activation of NOX/ROS/NF-κB signaling in MSCs contributes to the development of GONFH

doi: 10.1007/s10495-023-01860-2

Figure Lengend Snippet: DPI counteracted the OS-promoting effects of DEX and reversed apoptosis and imbalance in osteogenic/lipogenic differentiation of MSCs. ( A ) ROS staining and quantitative analysis of MSCs treated with DEX and DPI. ( B ) NOX2, NOX4, CASP3, OSX and PPARγ mRNA of MSCs treated with DEX and DPI. ( C ) NOX2, NOX4, CASP3, OSX and PPARγ protein levels of MSCs treated with DEX and DPI. ( D ) Tunel staining and flow cytometry of MSCs treated with DEX and DPI. ( E ) ALP and Oil red O staining of MSCs treated with DEX and DPI for 7 or 14 days. PC, positive control, * P < 0.05, ** P < 0.01, *** P < 0.001 (vs. DEX).

Article Snippet: Subsequently, sections were incubated with anti-RUNX2 (dilution 1:200, AB236639; Abcam, Cambridge, UK), anti-OSX (dilution 1:200, ER1914-47; Huabio, Hangzhou, Zhejiang, China), anti-PPARγ (dilution 1:300, AB19481; Abcam, Cambridge, UK), anti-fatty acid binding protein-4 (FABP4, dilution 1:300, AB92501; Abcam, Cambridge, UK), anti-NOX2 (dilution 1:500, 19013-1-AP; Proteintech, Chicago, USA), anti-NOX4 (dilution 1.

Techniques: Staining, TUNEL Assay, Flow Cytometry, Positive Control

Journal: Apoptosis

Article Title: Glucocorticoid-induced activation of NOX/ROS/NF-κB signaling in MSCs contributes to the development of GONFH

doi: 10.1007/s10495-023-01860-2

Figure Lengend Snippet: BAY partially reversed DEX-induced apoptosis and imbalance of osteogenic/lipogenic differentiation of MSCs. ( A ) Immunohistochemistry staining and quantitative analysis of p-p65 of rat femoral head. ( B ) NOX2, NOX4, CASP3, OSX and PPARγ mRNA of MSCs treated with DEX and BAY 11-7082. ( C ) CASP3, OSX and PPARγ protein levels of MSCs treated with DEX and BAY 11-7082. ( D ) ROS staining and quantitative analysis of MSCs treated with DEX and BAY 11-7082. ( E ) Tunel staining and flow cytometry of MSCs treated with DEX and BAY 11-7082. ( F ) ALP and Oil red O staining of MSCs treated with DEX and BAY 11-7082 for 7 or 14 days. PC, positive control, NS, no significance, * P < 0.05, ** P < 0.01, *** P < 0.001 (vs. GONFH or DEX).

Article Snippet: Subsequently, sections were incubated with anti-RUNX2 (dilution 1:200, AB236639; Abcam, Cambridge, UK), anti-OSX (dilution 1:200, ER1914-47; Huabio, Hangzhou, Zhejiang, China), anti-PPARγ (dilution 1:300, AB19481; Abcam, Cambridge, UK), anti-fatty acid binding protein-4 (FABP4, dilution 1:300, AB92501; Abcam, Cambridge, UK), anti-NOX2 (dilution 1:500, 19013-1-AP; Proteintech, Chicago, USA), anti-NOX4 (dilution 1.

Techniques: Immunohistochemistry, Staining, TUNEL Assay, Flow Cytometry, Positive Control

Journal: Molecular Biology Reports

Article Title: Synergistic effect of elevated glucose levels with SARS-CoV-2 spike protein induced NOX-dependent ROS production in endothelial cells

doi: 10.1007/s11033-023-08504-3

Figure Lengend Snippet: Protein expression in different doses of S protein induced HUVECs. A . ACE2 and TMPRSS2 protein expression relative to GAPDH analyzed by western blot in different doses of S protein induced HUVECs. B . Relative expression of gray values for ACE2. C . Relative expression of gray values for TMPRSS2. D . NOX1 protein expression relative to GAPDH analyzed by western blot in different doses of S protein induced HUVECs. E . Relative expression of gray values for NOX1. F . NOX2 protein expression relative to GAPDH analyzed by western blot in different doses of S protein induced HUVECs. G . Relative expression of gray values for NOX2. H . NOX3 protein expression relative to GAPDH analyzed by western blot in different doses of S protein induced HUVECs. I . Relative expression of gray values for NOX3. J . NOX4 protein expression relative to GAPDH analyzed by western blot in different doses of S protein induced HUVECs. K . Relative expression of gray values for NOX4

Article Snippet: The antibodies to ACE2 (#21115), TMPRSS2(#14437), NOX2(#19013), NOX4(#14347), and GAPDH (#60004) were obtained from Proteintech (Rosemont, IL).

Techniques: Expressing, Western Blot