Journal: Redox Biology

Article Title: SARS-CoV2 infection impairs the metabolism and redox function of cellular glutathione

doi: 10.1016/j.redox.2021.102041

Figure Lengend Snippet: Nrf2, Membrane transporters, and GCLC in SARS-CoV2 infected VERO-E6 cells treated with Nelfinavir (Nel) or Remdesivir (Rem). Immunoblot of Nrf2 protein expression ( A , left panels) was assessed 6 hpi and 24 hpi, and by semi-quantitative fluorescence analysis 48 hpi ( A , right panels). Fluorophores were FITC (green) for Nrf2 protein labelling, DAPI (blue) for nuclei and Phalloidin-Alexa Fluor595 (orange) for the cytosolic space. Immunoblot of xCT and MRP1 membrane transport proteins ( B ), and GCLC protein ( C ) were carried out as described in the section Methods 24 hpi. Infection conditions and treatments with antivirals were the same of . Control test with untreated cells (CTL -) vs infected cells or treatments: §p < 0.05, §§p < 0.01. Infected cells + DMSO vs antivirals *p < 0.05, **p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The primary antibodies used were: Nrf2 Polyclonal Antibody, FITC Conjugated (bs-1074R-FITC, Bioss Antibodies, 1:50); NQO1 (A180, Mouse mAb #3187, CST, 1:50); GST-pi (610,718, mouse mAb, BD Biosciences, 1:500); PE anti-human IL-6 Antibody (BioLegend, 1:1000); PE anti-human IL-10 Antibody (BioLegend, 1:1000).

Techniques: Infection, Western Blot, Expressing, Fluorescence

Journal: Bioengineered

Article Title: Long noncoding RNA BBOX1-AS1 promotes the progression of gastric cancer by regulating the miR-361-3p/Mucin 13 signaling axis.

doi: 10.1080/21655979.2022.2072629

Figure Lengend Snippet: Figure 5. MUC13 was targeted by miR-361-3p. (a) MUC13 and CLDN4 were predicted and screened as the target genes of miR-361- 3p using GEPIA and ENCORI. (b) RNA pull-down analysis on the interaction between MUC13 and CLDN4 of miR-361-3p. **P < 0.001 vs Bio-NC. (c) The binding sites of MUC13 on miR-361-3p were predicted by StarBase. (d) The luciferase reporter analysis on the relationship between miR-361-3p and MUC13. **P < 0.001 vs miR-NC. (e, f) The expression level of MUC13 in GC clinical samples (e) and GC cell lines (f) was detected by qRT-PCR. (g) Pearson analysis revealed the expression relationship between miR-361-3p and MUC13.

Article Snippet: These membranes were incubated with primary antibodies against GAPDH (1:1,000; #bs-10900 R; Bioss, Beijing, China) and MUC13 (1:1,000; #bs-1,074 R; Bioss, Beijing, China) at 4°C overnight and then incubated with the secondary antibody of horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5,000; #ab6721; Abcam) for 1 h at 25°C.

Techniques: Binding Assay, Luciferase, Expressing, Quantitative RT-PCR

Journal: Bioengineered

Article Title: Long noncoding RNA BBOX1-AS1 promotes the progression of gastric cancer by regulating the miR-361-3p/Mucin 13 signaling axis.

doi: 10.1080/21655979.2022.2072629

Figure Lengend Snippet: Figure 6. MiR-361-3p knockdown promoted GC progression by targeting MUC13. (a) The expression level of miR-361-3p was evaluated in groups of si-NC, inhibitor-NC, inhibitor, si-MUC13, and si-MUC13+ inhibitor by qRT-PCR. (b-d) The cell proliferation (b), invasion (c), and apoptosis (d) were measured in groups of si-NC, inhibitor-NC, inhibitor, si-MUC13, and si-MUC13+ inhibitor using CCK-8, Transwell and flow cytometry assays, respectively. **P < 0.001 vs si-NC, &&P < 0.001 vs inhibitor-NC. ##P < 0.001 vs si-lnc +inhibitor.

Article Snippet: These membranes were incubated with primary antibodies against GAPDH (1:1,000; #bs-10900 R; Bioss, Beijing, China) and MUC13 (1:1,000; #bs-1,074 R; Bioss, Beijing, China) at 4°C overnight and then incubated with the secondary antibody of horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5,000; #ab6721; Abcam) for 1 h at 25°C.

Techniques: Knockdown, Expressing, Quantitative RT-PCR, CCK-8 Assay, Flow Cytometry

Journal: Natural Product Communications

Article Title: Yougui Pills Alleviates Diminished Ovarian Reserve Through Regulating Oxidative Stress and Apoptosis in Rats

doi: 10.1177/1934578x241296409

Figure Lengend Snippet: Figure 4. YGP reduced oxidative stress by regulating Keap1/Nrf2. The protein levels of (A and B) Keap1, (A and C) Nrf2, (A and D) SOD2, (A and E) NQO1 and (A and F) NOX4 were determined using immunoblots. ##P < .01, ###P < .001 model group versus control group, *P < .05, **P < .01, ***P < .001 versus model group.YGP: Yougui pill.

Article Snippet: Enzyme-linked immunosorbent assay kits for FSH (MM-70867R1), LH (MM-0624R1), AMH (MM-0219R1), and Cortisol (MM-0574R1) were obtained from Wuhan Bioqiandu Technology Co., Ltd. Primary antibodies of Keap1 (1:1000, WL02135), Nrf2 (1:1000, WL02117), SOD2 (1:1000, WL02506), NQO1 (1:1000, WL04860), Bcl-2 (1:1000, WL01556), Bax (1:1000, WL01637), and Caspase3 (1:1000, WL02117), β-tubulin (1:1000, WL01931), and antirabbit secondary antibody (1:2000, WLA023) were purchased from Wanlei Biochemical Technology Co., Ltd. NOX4 (1:1000, 14347-1-AP) was purchased from proteintech.

Techniques: Western Blot, Control

Journal: Cell Death Discovery

Article Title: NSUN2 alleviates doxorubicin-induced myocardial injury through Nrf2-mediated antioxidant stress

doi: 10.1038/s41420-022-01294-w

Figure Lengend Snippet: The sequence of NSUN2 siRNA and Nrf2 primers.

Article Snippet: Rabbit NSUN2 (Proteintech, 20854-1-AP, 1:200) and Nrf2 (Proteintech, 16396-1-AP, 1:200) polyclonal antibodies were added and incubated at 37 °C for 2 h, and then the sections were incubated with goat anti-rabbit second antibody (ZSGO-BIO, PV-9001) at room temperature for 1 h. Finally, the results were tested by using the DAB Chromogenic Kit (ZSGO-BIO, ZLI-9018) according to the operating instructions.

Techniques: Sequencing

Journal: Cell Death Discovery

Article Title: NSUN2 alleviates doxorubicin-induced myocardial injury through Nrf2-mediated antioxidant stress

doi: 10.1038/s41420-022-01294-w

Figure Lengend Snippet: A , B Immunohistochemistry and western blot detected the expression level of Nrf2 in myocardial cells with and without DOX treatment (Bar = 100 µm). C IF detected the expression of Nrf2 and HO-1 after DOX-induced H9C2 cell injury (Bar = 20 µm). D IF detected the changes in intracellular ROS after DOX treatment. E , F Western blot detected the expression level of Nrf2 and HO-1 ( n = 3, Bar = 20 µm) after NSUN2 interference in H9C2 cells. G , H Western blot detected the effect of NSUN2 overexpression on the expression of Nrf2 and HO-1 in H9C2 cells injured by DOX. The measurement data were shown as mean ± standard deviation and compared by Student’s t -test. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Rabbit NSUN2 (Proteintech, 20854-1-AP, 1:200) and Nrf2 (Proteintech, 16396-1-AP, 1:200) polyclonal antibodies were added and incubated at 37 °C for 2 h, and then the sections were incubated with goat anti-rabbit second antibody (ZSGO-BIO, PV-9001) at room temperature for 1 h. Finally, the results were tested by using the DAB Chromogenic Kit (ZSGO-BIO, ZLI-9018) according to the operating instructions.

Techniques: Immunohistochemistry, Western Blot, Expressing, Over Expression, Standard Deviation

Journal: Cell Death Discovery

Article Title: NSUN2 alleviates doxorubicin-induced myocardial injury through Nrf2-mediated antioxidant stress

doi: 10.1038/s41420-022-01294-w

Figure Lengend Snippet: A IF detected the intracellular m5C level of H9C2 cells after DOX treatment ( n = 3, Bar = 20 μm). B Dot blot detected the changes of m5C methylation in H9C2 cells after DOX treatment. C IF detected the effect of NSUN2 overexpression on NSUN2 and Nrf2 expression levels and m5C enrichment in H9C2 cells (Bar = 20 μm). D m5C MeRIP-PCR detected the effect of NSUN2 overexpression on the enrichment of m5C in H9C2 cells ( n = 4). E RT-PCR detected the effect of NSUN2 overexpression on the half-life of Nrf2 mRNA ( n = 3). F m5C MeRIP test the content of m5C modified Nrf2 mRNA in NSUN2 and GFP group. G RT-PCR detected the effect of NSUN2 overexpression on the half-life of Nrf2 mRNA when treated with methylase inhibitor 3-DZA ( n = 3). The measurement data was showed as mean ± standard deviation, and compared by student’s t-test. ** P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: Rabbit NSUN2 (Proteintech, 20854-1-AP, 1:200) and Nrf2 (Proteintech, 16396-1-AP, 1:200) polyclonal antibodies were added and incubated at 37 °C for 2 h, and then the sections were incubated with goat anti-rabbit second antibody (ZSGO-BIO, PV-9001) at room temperature for 1 h. Finally, the results were tested by using the DAB Chromogenic Kit (ZSGO-BIO, ZLI-9018) according to the operating instructions.

Techniques: Dot Blot, Methylation, Over Expression, Expressing, Reverse Transcription Polymerase Chain Reaction, Modification, Standard Deviation

Journal: Cell Death Discovery

Article Title: NSUN2 alleviates doxorubicin-induced myocardial injury through Nrf2-mediated antioxidant stress

doi: 10.1038/s41420-022-01294-w

Figure Lengend Snippet: NSUN2 promote the methylation modification of Nrf2 mRNA, and increase the level of Nrf2 protein, which activates the downstream antioxidant stress response and reduces the damage caused by ROS.

Article Snippet: Rabbit NSUN2 (Proteintech, 20854-1-AP, 1:200) and Nrf2 (Proteintech, 16396-1-AP, 1:200) polyclonal antibodies were added and incubated at 37 °C for 2 h, and then the sections were incubated with goat anti-rabbit second antibody (ZSGO-BIO, PV-9001) at room temperature for 1 h. Finally, the results were tested by using the DAB Chromogenic Kit (ZSGO-BIO, ZLI-9018) according to the operating instructions.

Techniques: Methylation, Modification

Journal: PLoS ONE

Article Title: Electroacupuncture Ameliorates Acute Renal Injury in Lipopolysaccharide-Stimulated Rabbits via Induction of HO-1 through the PI3K/Akt/Nrf2 Pathways

doi: 10.1371/journal.pone.0141622

Figure Lengend Snippet: β-actin was monitored as the internal standard to ensure similar gel loading of the starting materials in each sample. Blot images were cropped for comparison (E). Electroacupuncture treatment significantly increased the levels of phospho-Akt protein, HO-1 protein, Nrf2 total and nucleoprotein compared with group L or group SEL. In wortmannin-treated rabbits, phosphorylation of Akt was significantly decreased while HO-1 protein, Nrf2 total and nucleoprotein was slightly decreased compared with group EL. All values were expressed as mean ±SD (n = 10; *P<0 . 05; **P<0 . 01 , using one-way ANOVA and the Bonferroni test for multiple comparisons). The blots were representative of three independent experiments.

Article Snippet: After protein blockade, the sections were incubated overnight at 4°Cwith the polyclonal Nrf2 primary antibody conjugated to FITC (dilution 1:150, Biorbyt, UK).

Techniques: Comparison, Phospho-proteomics

Journal: PLoS ONE

Article Title: Electroacupuncture Ameliorates Acute Renal Injury in Lipopolysaccharide-Stimulated Rabbits via Induction of HO-1 through the PI3K/Akt/Nrf2 Pathways

doi: 10.1371/journal.pone.0141622

Figure Lengend Snippet: (original magnification×400): (A) The pictures of immunofluorescence staining. Green stands for Nrf2-FITC stained sections, while blue stands for images of DAPI stained nuclei. The overlay color of blue staining in nucleus, accompanied with green staining both in cytoplasm and nucleus was considered to be positive. (B) Quantification of nuclear localization of Nrf2 protein among six groups. Electroacupuncture protocols dramatically increased translocation of Nrf2 from cytoplasm into nucleus compared with group L or SEL. To some degree, pretreatment with wortmannin counteracted nuclear accumulation of Nrf2 protein, while wortmannin alone had no effects. Data were representative of three independent experiments. Values were expressed as mean ± SD (n = 10; **P<0 . 01 , using one-way ANOVA and the Bonferroni test for multiple comparisons).

Article Snippet: After protein blockade, the sections were incubated overnight at 4°Cwith the polyclonal Nrf2 primary antibody conjugated to FITC (dilution 1:150, Biorbyt, UK).

Techniques: Immunofluorescence, Staining, Translocation Assay

Journal: Scientific Reports

Article Title: RNA sequencing of corneas from two keratoconus patient groups identifies potential biomarkers and decreased NRF2-antioxidant responses

doi: 10.1038/s41598-020-66735-x

Figure Lengend Snippet: Canonical pathways significantly altered in KC and KJ. Grey bars show total number of genes in the pathway (scale on top X axis), with blue and red bars showing the numbers of down-regulated and up-regulated genes (scale on bottom X axis). The p value next to each bar was calculated for that pathway by the IPA Core Analysis. “Acute Phase” is the Acute Phase Response Signaling pathway; “NRF2-mediated” is NRF2-mediated Oxidative Stress Response pathway; “ECM related” is the Hepatic Fibrosis / Hepatic Stellate Cell Activation pathway; “Rheumatoid Arthritis” is the Role of Macrophages, Fibroblasts and Endothelial Cells in Rheumatoid Arthritis pathway; and, “Osteoarthritis” includes genes associated with osteoarthritis.

Article Snippet: The following primary antibodies were used from Biorbyt Research Products: NRF2 (orb128433, 1:50), KEAP1 (orb48426, 1:100), Hsp40 (orb520080, 1:100), and GAS1 (orb414757, 1:100) and RXRA antibody were used from Novus Biologicals (NBP2–75653, 1:50).

Techniques: Activation Assay

Journal: Scientific Reports

Article Title: RNA sequencing of corneas from two keratoconus patient groups identifies potential biomarkers and decreased NRF2-antioxidant responses

doi: 10.1038/s41598-020-66735-x

Figure Lengend Snippet: NRF2 target genes significantly altered in KCN corneas. ( A ) Decreased NRF2 target gene expression in KC and KJ RNA seq. ( B ) Mechanism of NRF2 regulation by KEAP1; the latter binds to NRF2 and CUL3 for NRF2-ubiquitination and degradation to maintain low levels of NRF2 under homeostatic conditions. Under oxidative stress, KEAP1 dissociates from NRF2 allowing its increase and upregulation of target genes.

Article Snippet: The following primary antibodies were used from Biorbyt Research Products: NRF2 (orb128433, 1:50), KEAP1 (orb48426, 1:100), Hsp40 (orb520080, 1:100), and GAS1 (orb414757, 1:100) and RXRA antibody were used from Novus Biologicals (NBP2–75653, 1:50).

Techniques: Targeted Gene Expression, RNA Sequencing, Ubiquitin Proteomics

Journal: Scientific Reports

Article Title: RNA sequencing of corneas from two keratoconus patient groups identifies potential biomarkers and decreased NRF2-antioxidant responses

doi: 10.1038/s41598-020-66735-x

Figure Lengend Snippet: KEAP1 and NRF2 immunostaining in DN and KCN corneas. ( A ) KEAP1 staining is decreased in KCN corneas, with focally increased staining in some basal epithelial cells (inset), whereas in DN corneas KEAP1 shows staining of all epithelial layers (inset). ( B ) NRF2 shows very little to no staining of KCN corneas and these were all cytoplasmic (inset), while DN sections show stronger staining of epithelial cells and some nuclear staining (inset) DAPI nuclear staining shown in blue. IF staining of additional KCN and DN cornea sections are shown in Supplemental Fig. . Scale bar: 50 µm.

Article Snippet: The following primary antibodies were used from Biorbyt Research Products: NRF2 (orb128433, 1:50), KEAP1 (orb48426, 1:100), Hsp40 (orb520080, 1:100), and GAS1 (orb414757, 1:100) and RXRA antibody were used from Novus Biologicals (NBP2–75653, 1:50).

Techniques: Immunostaining, Staining