Journal: bioRxiv

Article Title: Alterations of redox and iron metabolism accompany development of HIV latency

doi: 10.1101/549014

Figure Lengend Snippet: (A) Subcellular localization of Nrf2 in CD4 + T-cells infected with HIV-1 or mock infected (3 dpi) as analyzed by biochemical fractionation. (B) Nuclear localization (left) and content (right) of Nrf2 in HIV-1 RNA + and HIV-1 RNA - cells (7 dpi) as measured by combining IF and HIV-1 RNA FISH. Scale bar = 2μm. n= number of cells from 3 donors. (C, D) Time course of the relative (infected vs mock infected) mRNA (C) and protein (D) levels of main targets of Nrf2 during the transition from productive (3-9 dpi) to latent (14 dpi) infection as measured by qPCR and western blot, respectively. TrxR1 and HMOX-1 were probed upon membrane stripping. (E) Comparison of the effect of different HIV-1 mutations on the mean relative (infected vs mock infected) mRNA level of the Nrf2 targets described in panel D. Values shown in panel B were calculated as nuclear corrected total cell fluorescence (as in) and analyzed by two-tailed unpaired t -test. For panels C, E, raw data were first normalized using GAPDH as housekeeping control and then expressed as Log 2 fold mRNA expression in infected vs mock infected cells (calculated using the 2-ΔΔCTmethod . In panel E the average of the six genes listed in Panel C is shown. For both panels data were analyzed by-two way ANOVA followed by Turkey’s post-test for multiple comparisons. ** P <0.01; *** P <0.001; **** P <0.001. Trx= thioredoxin; NQO1= NAD(P)H Quinone Dehydrogenase 1; HMOX-1= Heme Oxygenase 1; G6PD= glucose-6-phosphate dehydrogenase; GCLC= Glutamate—cysteine ligase; TrxR1= thioredoxin reductase 1.

Article Snippet: IF for Nrf2 (mab3925, 1:250; R&D systems, Minneapolis, MN) or PML (sc/966x, 1:500; Santa Cruz Biotechnology, Dallas, TX, USA) for 1-2 hr at RT.

Techniques: Infection, Fractionation, Western Blot, Membrane, Stripping Membranes, Comparison, Fluorescence, Two Tailed Test, Control, Expressing

Journal: Biomolecules

Article Title: BDH1 Mediates Aerobic Exercise-Induced Improvement in Skeletal Muscle Metabolic Remodeling in Type 2 Diabetes Mellitus

doi: 10.3390/biom16010115

Figure Lengend Snippet: Effects of AE on MQC and oxidative stress in the gastrocnemius muscle of WT T2DM mice. ( A ). Representative images of Fis1 immunofluorescence staining in the gastrocnemius muscle. Scale bars = 20 μm. ( B – D ). mRNA expression levels of genes regulating mitochondrial biogenesis (TFAM, PGC-1α, Nrf1) in the gastrocnemius muscle. n = 6. ( E , F ). mRNA expression levels of genes regulating mitochondrial fission (Drp1 and Fis1) in the gastrocnemius muscle. n = 6. ( G ). mRNA expression level of the gene regulating mitochondrial fusion (MFN2) in the gastrocnemius muscle. n = 6. ( H ). Nrf1, Drp1, MFN2, OPA1, and internal control β-actin protein expressions in the gastrocnemius muscle. ( I ). Nrf1 protein level quantitative analysis. n = 3. ( J ). Drp1 protein level quantitative analysis. n = 3. ( K ). MFN2 protein level quantitative analysis. n = 3. ( L ). OPA1 protein level quantitative analysis. n = 3. ( M – O ). Antioxidant (Nrf2, Gpx4, and SOD2) mRNA expression levels in the gastrocnemius muscle. n = 6. ( P ). Nrf2 and internal control β-actin protein expressions in the gastrocnemius muscle. ( Q ). Nrf2 protein level quantitative analysis. n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001. Original images of ( H , P ) can be found in .

Article Snippet: The primary antibodies used in this study were as follows: α-SMA (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA, sc-53142), DRP1 (1:1000, Cell Signaling Technology, Boston, MA, USA, 8570S), MFN2 (1:1000, Cell Signaling Technology, Boston, MA, USA, 9482S), p-Akt (1:1000, Cell Signaling Technology, Boston, MA, USA, 4060S), OPA1 (1:1000, Cell Signaling Technology, Boston, MA, USA, 80471S), Glut4 (1:1000, Cell Signaling Technology, Boston, MA, USA, 2213S), PI3K (1:1000, Cell Signaling Technology, Boston, MA, USA, 4249S), BDH1 (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA, sc-514413), Nrf1 (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA, sc-515360), Nrf2 (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA, sc-365949), and β-actin (1:20000, Proteintech, Wuhan, China, 66009-1-Ig).

Techniques: Immunofluorescence, Staining, Expressing, Control

Journal: Biomolecules

Article Title: BDH1 Mediates Aerobic Exercise-Induced Improvement in Skeletal Muscle Metabolic Remodeling in Type 2 Diabetes Mellitus

doi: 10.3390/biom16010115

Figure Lengend Snippet: Effects of AE on MQC and oxidative stress in the gastrocnemius muscle of BDH1 −/− T2DM mice. ( A ). BDH1 mRNA expression level in the gastrocnemius muscle. n = 6. ( B ). Protein expression of BDH1 and internal control β-actin in the gastrocnemius muscle. ( C ). BDH1 protein level quantitative analysis. n = 3. ( D ). Representative images of Fis1 immunofluorescence staining in the gastrocnemius muscle. Scale bars = 20 μm. ( E – G ). mRNA expression levels of genes regulating mitochondrial biogenesis (TFAM, PGC-1α, and Nrf1) in the gastrocnemius muscle. n = 6. ( H , I ). mRNA expression levels of genes regulating mitochondrial fission (Drp1 and Fis1) in the gastrocnemius muscle. n = 6. ( J ). mRNA expression level of the gene regulating mitochondrial fusion (MFN2) in the gastrocnemius muscle. n = 6. ( K ). Protein expression of Nrf1, Drp1, MFN2, OPA1, and internal control β-actin in the gastrocnemius muscle. ( L ). Nrf1 protein level quantitative analysis. n = 3. ( M ). Drp1 protein level quantitative analysis. n = 3. ( N ). MFN2 protein level quantitative analysis. n = 3. ( O ). OPA1 protein level quantitative analysis. n = 3. ( P – R ). Antioxidant (Nrf2, Gpx4, SOD2) mRNA expression levels in the gastrocnemius muscle. n = 6. ( S ). Nrf2 and internal control β-actin protein expressions in the gastrocnemius muscle. ( T ). Nrf2 protein level quantitative analysis. n = 3. * p < 0.05, ** p < 0.01, *** p < 0.001. Original images of ( B , K , S ) can be found in .

Article Snippet: The primary antibodies used in this study were as follows: α-SMA (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA, sc-53142), DRP1 (1:1000, Cell Signaling Technology, Boston, MA, USA, 8570S), MFN2 (1:1000, Cell Signaling Technology, Boston, MA, USA, 9482S), p-Akt (1:1000, Cell Signaling Technology, Boston, MA, USA, 4060S), OPA1 (1:1000, Cell Signaling Technology, Boston, MA, USA, 80471S), Glut4 (1:1000, Cell Signaling Technology, Boston, MA, USA, 2213S), PI3K (1:1000, Cell Signaling Technology, Boston, MA, USA, 4249S), BDH1 (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA, sc-514413), Nrf1 (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA, sc-515360), Nrf2 (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA, sc-365949), and β-actin (1:20000, Proteintech, Wuhan, China, 66009-1-Ig).

Techniques: Expressing, Control, Immunofluorescence, Staining

Journal: NPJ Science of Food

Article Title: From rosemary and coffee to bioactive nanovesicles: exploring new frontiers in food functional ingredients

doi: 10.1038/s41538-026-00723-9

Figure Lengend Snippet: Nrf2 ( A ) and COX-2 ( B ) protein levels in C2C12 cells pretreated with RVs and CVs 39 × 10 6 vesicles/mL in oxidative stress-induced damage. The data points represent the averages ±SD of three independent experiments in duplicate. All datasets were analyzed by one-way ANOVA followed by Tukey’s post hoc test. Different lowercase letters indicate a significant difference ( p 0.05) between different treatments. Control: untreated cells. RVs rosemary-derived vesicles, CVs coffee-derived vesicles. 7.8 × 10 6 vesicles/mL and 39 × 10 6 vesicles/mL correspond to 0.1 and 0.5 mg nanovesicles/mL.

Article Snippet: The antibody against Collagen Type I (Cat No. 66761-1-Ig) and Fatty Acis Synthase (FASN, Cat No. 10624-2-AP), COX2/Cyclooxygenase 2 (Cat No. 12375-1-AP) and nuclear factor erythroid 2-related factor 2 (NRF2) (Cat No. 66504-1-Ig) were from Proteintech.

Techniques: Control, Derivative Assay

Journal: The Journal of nutrition

Article Title: Ajoene, a stable garlic by-product, has an antioxidant effect through Nrf2-mediated glutamate-cysteine ligase induction in HepG2 cells and primary hepatocytes.

doi: 10.3945/jn.110.121277

Figure Lengend Snippet: FIGURE 1 Activation of Nrf2 by ajoene in HepG2 cells treated with 3–30 mmol/L ajoene or 30 mmol/L t-BHQ for 6 h (A) or 30 mmol/L ajoene for 3–12 h (B) and ARE reporter gene activity in HepG2 cells treated with 3–30 mmol/L ajoene or 30 mmol/L t-BHQ for 24 h after transfection of the NQO1-ARE luciferase construct. Values are means 6 SD, n = 4. Means without a common letter differ, P , 0.05.

Article Snippet: Antibodies directed against phospho-PKCd, phospho-extracellular signal-regulated kinase (ERK) (Thr202/Tyr204), phospho-c-Jun NH2-terminal kinase (JNK) (Thr183/ Tyr185), phospho-Akt, and lamin A/C were obtained from Cell Signaling Technology, whereas that of phospho-Nrf2 (Ser40) was supplied from Novus Biologicals.

Techniques: Activation Assay, Activity Assay, Transfection, Luciferase, Construct

Journal: The Journal of nutrition

Article Title: Ajoene, a stable garlic by-product, has an antioxidant effect through Nrf2-mediated glutamate-cysteine ligase induction in HepG2 cells and primary hepatocytes.

doi: 10.3945/jn.110.121277

Figure Lengend Snippet: FIGURE 3 Inhibition of Nrf2 ubiquitination by ajoene in HepG2 cells. (A) The interaction between Keap1 and Nrf2. Immunoprecipitated Nrf2 was subjected to immunoblotting for Keap1. Results were confirmed in 3 replicate experiments. (B) Nrf2 ubiquitination. Cells were transfected with the plasmid encoding His-tagged ubiquitin (His-Ubi), treated with 10 mmol/L MG132 (a proteasomal inhibitor) for 1 h, and continuously incubated with 30 mmol/L ajoene or 30 mmol/L t-BHQ for 6 h. Immunoprecipitated His-Ubi was subjected to immunoblottings for Nrf2. Mock transfection of pcDNA3 (an empty vector) was used as control. Values are means 6 SD, n = 4. Means without a common letter differ, P , 0.05. IP, Immunoprecipitation; IB, immunoblotting.

Article Snippet: Antibodies directed against phospho-PKCd, phospho-extracellular signal-regulated kinase (ERK) (Thr202/Tyr204), phospho-c-Jun NH2-terminal kinase (JNK) (Thr183/ Tyr185), phospho-Akt, and lamin A/C were obtained from Cell Signaling Technology, whereas that of phospho-Nrf2 (Ser40) was supplied from Novus Biologicals.

Techniques: Inhibition, Ubiquitin Proteomics, Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation, Incubation, Control

Journal: The Journal of nutrition

Article Title: Ajoene, a stable garlic by-product, has an antioxidant effect through Nrf2-mediated glutamate-cysteine ligase induction in HepG2 cells and primary hepatocytes.

doi: 10.3945/jn.110.121277

Figure Lengend Snippet: FIGURE 4 PKCd-dependent increases in GCLC and GCLM mRNA levels in HepG2 cells. (A) The effects of ajoene on the phosphorylation of upstream kinases. Immunoblot analyses were performed on cell lysates that had been incubated with 30 mmol/L ajoene for 10 min to 6 h. (B) Inhibition of Nrf2 phosphorylation and nuclear accumulation by rottlerin. Cells were treated with 30 mmol/L ajoene alone or in combination with 2 mmol/L rottlerin or 5 mmol/L U73122 for 6 h. Results were confirmed in 3 replicate experiments. GCLC (C) and GCLM (D) mRNA levels measured using real-time PCR assays in cells that had been treated as described in B. Values are means 6 SD, n = 4. Means without a common letter differ, P , 0.05.

Article Snippet: Antibodies directed against phospho-PKCd, phospho-extracellular signal-regulated kinase (ERK) (Thr202/Tyr204), phospho-c-Jun NH2-terminal kinase (JNK) (Thr183/ Tyr185), phospho-Akt, and lamin A/C were obtained from Cell Signaling Technology, whereas that of phospho-Nrf2 (Ser40) was supplied from Novus Biologicals.

Techniques: Phospho-proteomics, Western Blot, Incubation, Inhibition, Real-time Polymerase Chain Reaction

Journal: The Journal of nutrition

Article Title: Ajoene, a stable garlic by-product, has an antioxidant effect through Nrf2-mediated glutamate-cysteine ligase induction in HepG2 cells and primary hepatocytes.

doi: 10.3945/jn.110.121277

Figure Lengend Snippet: FIGURE 5 Nrf2-dependent antioxidant capacity in HepG2 cells (A,B) or primary murine hepatocytes (C). (A) The GSH concentration was measured in lysates of cells treated with 300 mmol/L t-BHP and/ or 30 mmol/L ajoene. (B) Viability of HepG2 cells treated with 3–30 mmol/L ajoene for 12 h and continuously incubated with 300 mmol/L t-BHP for 12 h. (C) DCFH oxidation in primary hepatocytes from Nrf2 gene knockout (Nrf22/2) and wildtype (WT) mice exposed to ajoene and/or t-BHP as described above. Values are means 6 SD, n = 4. Means without a common letter differ, P , 0.05.

Article Snippet: Antibodies directed against phospho-PKCd, phospho-extracellular signal-regulated kinase (ERK) (Thr202/Tyr204), phospho-c-Jun NH2-terminal kinase (JNK) (Thr183/ Tyr185), phospho-Akt, and lamin A/C were obtained from Cell Signaling Technology, whereas that of phospho-Nrf2 (Ser40) was supplied from Novus Biologicals.

Techniques: Concentration Assay, Incubation, Gene Knockout

Journal: The Journal of nutrition

Article Title: Ajoene, a stable garlic by-product, has an antioxidant effect through Nrf2-mediated glutamate-cysteine ligase induction in HepG2 cells and primary hepatocytes.

doi: 10.3945/jn.110.121277

Figure Lengend Snippet: FIGURE 6 A schematic diagram illustrating the proposed mechanism by which ajoene protects cells against oxidative stress. Ajoene activates PKCd, which leads to Nrf2 activation. Ajoene inhibits interaction between Keap1 and Nrf2, thereby repressing Nrf2 ubiquitination, as did t-BHQ. Nrf2 activation by ajoene contributes to GCLC and GCLM induction and restoration of cellular GSH concentra- tion, which antagonizes oxidative stress.

Article Snippet: Antibodies directed against phospho-PKCd, phospho-extracellular signal-regulated kinase (ERK) (Thr202/Tyr204), phospho-c-Jun NH2-terminal kinase (JNK) (Thr183/ Tyr185), phospho-Akt, and lamin A/C were obtained from Cell Signaling Technology, whereas that of phospho-Nrf2 (Ser40) was supplied from Novus Biologicals.

Techniques: Activation Assay, Ubiquitin Proteomics