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

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Soluble Epoxide Hydrolase Inhibition Protected against Diabetic Cardiomyopathy through Inducing Autophagy and Reducing Apoptosis Relying on Nrf2 Upregulation and Transcription Activation

doi: 10.1155/2022/3773415

Figure Lengend Snippet: AUDA administration alleviated cardiac dysfunction and reduced apoptosis in db/db mice heart. (a) Representative immunoblots and quantitation of soluble epoxide hydrolase (sEH) expression, (b) +dp/dt max, (c) -dp/dt min, (d) left ventricular ejection fraction (LVEF), (e) left ventricular internal diameter end systole (LVID(s)), (f) left ventricular internal diameter end diastole (LVID(d)), and (g) E/A ratio. (h and i) Representative immunoblots and quantitation of protein expression of cleaved caspase-3, Bax, Bcl-2, NOX4, NOX2, and SOD1 in the myocardia of mice treated with or without the sEH inhibitor AUDA. (j and k) Representative immunoblots and quantitation of protein expression of cleaved caspase-3, Bax, Bcl-2, NOX4, NOX2, and SOD1 in HGHF-stimulated cardiomyocytes pretreated with or without AUDA. (l–o) Histological analysis and quantitation of DHE, TUNEL, and 3-NT staining in the myocardia in different groups. Data were expressed as mean ± SEM ( n = 8 mice per group). ∗ p < 0.05 vs. Con and # p < 0.05 vs. DM or HGHF. Con: db/m+vehicle; AUDA: db/m+AUDA; DM: db/db+vehicle; MABSA: mannitol+ BSA; DHE: dihydroethidium; HGHF: high glucose and high fat.

Article Snippet: The following antibodies were used: Bax (BosterBio, Pleasanton, CA, USA), Bcl2 (Boster), caspase-3 (Proteintech, Rosemont, IL, USA), cleaved caspase-3 (Cell Signaling Technology (CST), Danvers, MA, USA), NOX2 (Proteintech), NOX4 (Proteintech), SOD1 (Proteintech), Beclin 1 (CST), Atg3 (Santa Cruz Biotechnology, Dallas, TX, USA), LC3II (CST), and β -actin (Santa Cruz Biotechnology).

Techniques: Western Blot, Quantitation Assay, Expressing, TUNEL Assay, Staining

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Soluble Epoxide Hydrolase Inhibition Protected against Diabetic Cardiomyopathy through Inducing Autophagy and Reducing Apoptosis Relying on Nrf2 Upregulation and Transcription Activation

doi: 10.1155/2022/3773415

Figure Lengend Snippet: Small interfering RNA of Nrf2 specifically reduced the protective effects of AUDA on cardiomyocytes autophagy and apoptosis. (a and b) Representative immunoblots and quantitation of protein expression of Beclin 1, Atg3, and LC3II in HGHF-stimulated cardiomyocytes in different groups. (c) LC3-II/ β -actin ratio with or without bafilomycin A1 treatment under HGHF culture conditions. (d and e) Representative images of fluorescent LC3 puncta and quantitation of cardiomyocytes with different treatments. (f and g) Representative immunoblots and quantitation of protein expression of cleaved caspase-3, Bax, Bcl-2, NOX4, NOX2, and SOD1 in the HGHF-stimulated cardiomyocytes pretreated with or without AUDA. (h and i) Representative images and quantitation of cardiomyocyte apoptosis via the Annexin-V/PI apoptotic assay. (j and k) Representative images and quantitation of cardiomyocyte ROS levels via DCFH-DA. Data were expressed as mean ± SEM (n ≥ 3 per group). ∗ p < 0.05 vs. si-Con+Con and # p < 0.05 vs. si-Nrf2+Con. HGHF: high glucose and high fat.

Article Snippet: The following antibodies were used: Bax (BosterBio, Pleasanton, CA, USA), Bcl2 (Boster), caspase-3 (Proteintech, Rosemont, IL, USA), cleaved caspase-3 (Cell Signaling Technology (CST), Danvers, MA, USA), NOX2 (Proteintech), NOX4 (Proteintech), SOD1 (Proteintech), Beclin 1 (CST), Atg3 (Santa Cruz Biotechnology, Dallas, TX, USA), LC3II (CST), and β -actin (Santa Cruz Biotechnology).

Techniques: Small Interfering RNA, Western Blot, Quantitation Assay, Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Soluble Epoxide Hydrolase Inhibition Protected against Diabetic Cardiomyopathy through Inducing Autophagy and Reducing Apoptosis Relying on Nrf2 Upregulation and Transcription Activation

doi: 10.1155/2022/3773415

Figure Lengend Snippet: Virus carrying code for Nrf2 shRNA specifically reduced the protective effects of AUDA on apoptosis in db/db mice heart. (a and b) Representative immunoblots and quantitation of protein expression of cleaved caspase-3, Bax, Bcl-2, NOX4, NOX2, and SOD1 in the myocardia of mice from different groups. (c–f) Histological analysis and quantitation of DHE, TUNEL, and 3-NT staining in the myocardia from different groups. (g) Mechanism diagram of full text. Data were expressed as mean ± SEM (n = 8 per group). ∗ p < 0.05 vs. ScshRNA +Con, # p < 0.05 vs. Nrf2shRNA+Con, and & p < 0.05 vs. Nrf2shRNA+DM. ScshRNA: AAV-ScshRNA; Nrf2shRNA: AAV-Nrf2shRNA; Con: db/m+vehicle; AUDA: db/m+AUDA; DM: db/db+vehicle; DHE: dihydroethidium.

Article Snippet: The following antibodies were used: Bax (BosterBio, Pleasanton, CA, USA), Bcl2 (Boster), caspase-3 (Proteintech, Rosemont, IL, USA), cleaved caspase-3 (Cell Signaling Technology (CST), Danvers, MA, USA), NOX2 (Proteintech), NOX4 (Proteintech), SOD1 (Proteintech), Beclin 1 (CST), Atg3 (Santa Cruz Biotechnology, Dallas, TX, USA), LC3II (CST), and β -actin (Santa Cruz Biotechnology).

Techniques: shRNA, Western Blot, Quantitation Assay, Expressing, TUNEL Assay, Staining

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Angiotensin II Induces Cardiac Edema and Hypertrophic Remodeling through Lymphatic-Dependent Mechanisms

doi: 10.1155/2022/5044046

Figure Lengend Snippet: VEGFR-3 knockdown accelerates Ang II-induced cardiac fibrosis, oxidative stress, and inflammation in mice. VEGFR-3 f/f and Lyve-1 Cre VEGFR-3 f/− mice were infused with saline or Ang II (1000 ng/kg/min) for 14 days. (a) Cardiac Masson staining (left) and quantitative analysis of the fibrotic area (%) (right, n = 6). (b) qPCR analyses of myocardial collagen I and collagen III levels ( n = 6). (c) Cardiac α -SMA immunohistochemical staining (left) and quantitative analysis of the positive area (right, n = 6). (d) qPCR analyses of myocardial α -SMA levels ( n = 6). (e) Cardiac DHE fluorescence staining (left) and quantitative analysis of relative fluorescence intensity (right, n = 6). (f) qPCR analyses of myocardial NOX2 and NOX4 levels ( n = 6). (g) CD68 (red) and DAPI (blue) fluorescence staining of heart sections (left) and quantification of CD68 + macrophages (right, n = 6). (h) qPCR analyses of myocardial IL-1 β and IL-6 levels ( n = 6). (i) Immunoblot analysis of cardiac TGF- β 1, p-Smad2/3, Smad2/3, NOX2, NOX4, p-P65, and P65 levels (left) and quantification (right, n = 4). The data are presented as the mean ± SD; n represents the number of animals in each group. Statistical differences were determined by two-way ANOVA; ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 versus the VEGFR-3 f/f +saline group; # p < 0.05, ## p < 0.01, and ### p < 0.001 versus the VEGFR-3 f/f +Ang II group.

Article Snippet: The following antibodies were used: anti-VEGF-C (22601-1-AP, Proteintech), anti-VEGFR-3 (ab273148, Abcam), anti-p-p38 (ARG51850, Arigo), anti-p38 (ARG55258, Arigo), anti-VE-cadherin (ab205336, Abcam), anti-p-ERK1/2 (4370, CST), anti-ERK1/2 (4695S, CST), anti-p-AKT (9271S, CST), anti-AKT (9272, CST), anticalcineurin A (CaNA) (2614, CST), anti-p-STAT3 (9145, CST), anti-STAT3 (9139, CST), anti-TGF- β 1 (3711, CST), anti-p-Smad2/3 (882S, CST), anti-Smad2/3 (8685, CST), anti-NOX2 (19013-1-AP, Proteintech), anti-NOX4 (14347-1-AP, Proteintech), anti-p-P65 (3033, CST), anti-P65 (4764, CST), anti- β 2i (13635, CST), anti- β 5i (ARG41022, Arigo) anti-MKP5 (374276, Santa), and anti-GAPDH (5174, CST).

Techniques: Staining, Immunohistochemical staining, Fluorescence, Western Blot

Journal: Asian Journal of Andrology

Article Title: Lipoxin A4 improves erectile dysfunction in rats with type I diabetes by inhibiting oxidative stress and corporal fibrosis

doi: 10.4103/aja.aja_49_17

Figure Lengend Snippet: Treatment of LXA4 inhibits oxidative stress in corpus cavernous of DMED rats. ( a ) Representative Western blot results for the p22 phox , p47 phox , gp91 phox , p40 phox , and p67 phox in rats of all the three groups. ( b ) Expressions of p22 phox , p47 phox , gp91 phox , p40 phox , and p67 phox with β-actin as the loading control in all the three groups were presented through bar graphs. ( c ) MDA levels determined by the ELISA method in all the three groups. ( d ) SOD activities determined by the ELISA method in all the three groups. Data are expressed as mean ± s.e.m. ( n control = 10, n DMED = 11, and n DMED + LXA4 = 11). * P < 0.05 and *** P < 0.001 when comparing two groups. DMED: diabetes mellitus-induced erectile dysfunction; LXA4: Lipoxin A4; ELISA: enzyme-linked immunosorbent assay; SOD: superoxide dismutase; MDA: malondialdehyde; s.e.m.: standard error of the mean.

Article Snippet: Equal amounts of protein samples (30 μg per lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes, followed by blocking in Tris-buffered saline-Tween (TBST) with 5% bovine serum albumin (BSA) for 1 h. Then, membranes were incubated with primary antibodies against: eNOS (1:1000; ab5589, Abcam, Cambridge, MA, USA), p-eNOS (Ser1177; 1:1000; 9571; Cell Signaling Technology, Danvers, MA, USA), neuronal nitric oxide synthase (nNOS; 1:1000; ab76067; Abcam), p22 phox (1:500; sc-271968, Santa Cruz, Dallas, Texas, USA), p47 phox (1:1000; D154042, Sangon Biotech, Shanghai, China), gp91 phox (1:500, 19013-1-AP, Proteintech); p40 phox (1:500; D154073, Sangon Biotech), p67 phox (1:1000; 15551-1-AP, Proteintech), α-smooth muscle actin (α-SMA; 1:1000; ab5694; Abcam), Collagen I (1:500; PB0981, Boster, Wuhan, China), Collagen IV (1:500; 19797-1-AP, Proteintech), TGF-β1 (1:1000; ab92486, Abcam), Smad2/3 (1:1000; 8685, Cell Signaling Technology), p-Smad2/3 (1:1000; 8828, Cell Signaling Technology), CTGF (1:1000; 23936-1-AP, Proteintech), and β-actin (1:1000, 20536-1-AP, Proteintech).

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Downregulated Expression of Solute Carrier Family 26 Member 6 in NRK-52E Cells Attenuates Oxalate-Induced Intracellular Oxidative Stress

doi: 10.1155/2018/1724648

Figure Lengend Snippet: Higher Slc26a6 expression induced more intracellular ROS generation by oxalate. (a) Four cell types were stimulated with oxalate (700 μ M) for 3 h, and intracellular ROS levels were determined by fluorescence intensity of DCFH analyzed using flow cytometry. Changes before and after oxalate treatment are shown on the right graph as means ± SD ( n = 3). ∗ P < 0.05. (b) Lipid peroxidation was assessed by detecting the MDA level in supernatants of NRK cell lysates. NRK-Slc26a6 cells showed an obvious increase in MDA generation compared with control groups after oxalate treatment ( n = 6). (c) Activity change of superoxide dismutase (SOD) expressed as means ± SD. In the NRK-Slc26a6 group, SOD activity was markedly decreased compared to that in control groups. (d, e, f) Expressions of NADPH oxidase 2 (Nox2) and Nox4 before and after oxalate treatment in all groups using Western blot analysis. After oxalate treatment (700 μ M) for 24 h, the expression of Nox4 increased in the NRK-Slc26a6 group and that of Nox2 was decreased in the NRK-siRNA group. Data are means ± SD ( n = 3), ∗ P < 0.05.

Article Snippet: The PVDF membranes were blocked with 5% bovine serum albumin (BSA) for 2 h and probed with primary antibodies against Slc26a6 (1 : 200, sc-26728, Santa Cruz Biotechnology, CA, USA), NADPH oxidase 2 (Nox2, 1 : 200, 19013-1-AP, Proteintech, Hubei, China), Nox4 (1 : 200, 14347-1-AP, Proteintech, Hubei, China), NF κ B p65 (1 : 500, GB11142, Servicebio, Hubei, China), NF κ B-p65 (phosphorylated [p]-Ser536, 1 : 500, 11014, Signalway Antibody Co., Ltd, MD, USA), I κ B α (1 : 500, GB13212-1, Servicebio, Hubei, China), I κ B α (p-Ser32/36, 1 : 500, 11152, Signalway Antibody Co., Ltd, MD, USA), and OPN (1 : 200, 22952-1-AP, Proteintech, Hubei, China) at 4°C overnight.

Techniques: Expressing, Fluorescence, Flow Cytometry, Activity Assay, Western Blot