Journal: Journal of Advanced Research

Article Title: Long non-coding RNA LINC01532 sustains redox homeostasis and accelerates lenvatinib resistance in hepatocellular carcinoma

doi: 10.1016/j.jare.2025.02.035

Figure Lengend Snippet: LINC01532 is identified as a potential NADPH-metabolism-related lncRNA in HCC drug resistance. ( A ) The expression of G6PD, ME1, ME2 and MTHFD2 was analyzed in HCC tissues and adjacent non-cancerous tissues using TCGA dataset. ( B ) Venn diagram was employed to analyze the overlap lncRNAs among G6PD-related lncRNAs, prognosis related lncRNAs and differentially expressed lncRNAs. ( C ) The expression of the eight lncRNAs was evaluated by RT-qPCR in HCC tissues and the adjacent non-tumoral tissues from our hospital. ( D ) HuH-7 cells were transfected with indicated siRNAs and cellular NADPH level was determined. ( E ) The expression of eight lncRNAs was evaluated by RT-qPCR in lenvatinib-resistant HuH-7 cells. ( F ) The LINC01532 expression was analyzed in HCC tissues and adjacent non-tumoral tissues. ( G ) Overall survival (OS) and disease-free survival (DFS) analyses based on LINC01532 levels were analyzed using data from TCGA-LIHC cohort. ( H ) OS and DFS analyses based on LINC01532 level were analyzed using data from our cohort. ( I ) RT-qPCR was performed to detect LINC01532 expression in the cytoplasmic and nuclear fractions. ( J ) The subcellular localization of LINC01532 was determined by Fluorescence in situ hybridization (FISH). Scale bar, 10 μm. ( K ) LINC01532 coding capability was performed by CPAT ( http://lilab.research.bcm.edu ).

Article Snippet: NADPH, G6PD, GSH and MDA levels were evaluated using NADP + /NADPH Assay Kit (Beyotime, China), G6PDH Activity Assay Kit (Beyotime, China), Reduced Glutathione Content Assay Kit (Solarbio, China) and Malondialdehyde (MDA) Colorimetric Assay Kit (Elabscience, China), respectively, based on the manufacturer's protocol.

Techniques: Expressing, Quantitative RT-PCR, Transfection, Fluorescence, In Situ Hybridization

Journal: Journal of Advanced Research

Article Title: Long non-coding RNA LINC01532 sustains redox homeostasis and accelerates lenvatinib resistance in hepatocellular carcinoma

doi: 10.1016/j.jare.2025.02.035

Figure Lengend Snippet: LINC01532 elevates PPP flux. ( A ) Cellular NADPH level was determined in HuH-7 and MHCC97-H stably overexpressing LINC01532. ( B ) Cellular G6PD activity was evaluated in the indicated cells. ( C ) Cellular NADPH level was detected in the indicated cells. ( D ) Cellular G6PD activity was determined in the indicated cells. ( E ) Cellular GSH level was assessed in HuH-7 and MHCC97-H stably overexpressing LINC01532. ( F ) Cellular GSH level was analyzed in the indicated cells. ( G ) Oil red O staining assay (left) was performed in the indicated cells. The numbers of lipid droplets around cells were summarized (n = 50, right). ( H ) Cellular NADPH level was determined in the indicated cells treated with RRX-001. ( I ) The indicated cells were treated with RRX-001 and cellular GSH level was determined. ( J ) Cellular NADPH level was determined in the cells transfected as indicated. ( K ) Oil red O staining (left) and statistical analysis (right) were performed in HuH-7 cells as indicated.

Article Snippet: NADPH, G6PD, GSH and MDA levels were evaluated using NADP + /NADPH Assay Kit (Beyotime, China), G6PDH Activity Assay Kit (Beyotime, China), Reduced Glutathione Content Assay Kit (Solarbio, China) and Malondialdehyde (MDA) Colorimetric Assay Kit (Elabscience, China), respectively, based on the manufacturer's protocol.

Techniques: Stable Transfection, Activity Assay, Staining, Transfection

Journal: Redox Report : Communications in Free Radical Research

Article Title: Urolithin A alleviates vascular remodeling through mitochondrial SIRT3-mediated SOD2 deacetylation and antioxidation in hypertensive rats

doi: 10.1080/13510002.2026.2622255

Figure Lengend Snippet: Effects of UA on oxidative stress in VSMCs of WKY and SHR. A, dose-effect and time effect of UA on superoxide production in VSMCs. B, NADPH oxidase (NOX) activity. C, NOX1/2/4 protein expressions. D, relative DCF fluorescence intensity (green) showing intracellular ROS levels. E, relative mitoSOX Red fluorescence intensity (red) showing mitochondrial ROS levels. Nuclei were stained with Hoechst (blue). Values are mean ± SD. * P < 0.05 vs 0 μM or 0 h; † P < 0.05 vs PBS or DMSO; # P < 0.05 vs WKY. n = 6 for A-B & D-E; n = 4 for C. Two-way ANOVA followed by Bonferroni post hoc test.

Article Snippet: Antibodies against NOX1 (Cat. #17772-1-AP), NOX2 (Cat. #19013-1-AP), NOX4 (Cat. #14347-1-AP), SOD2 (Cat. #24127-1-AP), COXIV (Cat. #11242-1-AP), PCNA (Cat. #10205-2-AP) and β-actin (Cat. #66009-1-Ig) were bought from Proteintech Group, Inc. (Chicago, IL, USA).

Techniques: Activity Assay, Fluorescence, Staining

Journal: Journal of molecular and cellular cardiology

Article Title: Netrin-1 abrogates ischemia/reperfusion-induced cardiac mitochondrial dysfunction via nitric oxide-dependent attenuation of NOX4 activation and recoupling of NOS.

doi: 10.1016/j.yjmcc.2014.07.005

Figure Lengend Snippet: Fig. 1. Netrin-1 attenuates I/R induced increases in superoxide production, NOX activity, NOX4 protein abundance and mitochondrial dysfunction. A) Schematic illustration of experimen- tal protocols. B) Superoxide production from I/R injured hearts with and without netrin-1 perfusion. Superoxide production from heart homogenates was measured using electron spin resonance (ESR), shown as the amount inhibited by Mn-SOD, and normalized to no I/R condition. Superoxide production from the I/R control was significantly higher than all other conditions (p = 0.001 vs. all others, n = 3). Of note, the 2.5-fold increase in superoxide production provoked by I/R was completely attenuated by netrin-1 perfusion. C) NADPH- driven NOX activity assessed using purified membrane faction of perfused hearts. Measurements shown are superoxide production under control (without addition of NADPH substrate, top), with NADPH (middle), and the difference between the two (bottom), which is an indication of NOX activity. Data indicate that under I/R, NOX activity was significantly increased compared to no I/R, which was completely attenuated by netrin-1 perfusion (p b 0.001 vs. all others, n = 4). D) Western blot showing the specificity of the NOX4 antibody used. NOX4 protein expression was increased in the plasmid overexpressed cells, while decreased in the NOX4 siRNA treated cells. E) Protein levels of NOX1, NOX2 and NOX4 in no I/R, I/R, and netrin-1 perfused I/R-injured hearts. NOX1 and NOX2 protein levels were unchanged. NOX4 was significantly and reproducibly upregulated in I/R-injured hearts, which was abolished by netrin-1 perfusion (p b 0.001 vs. all others, n = 4). F) NOX4 activity assessed using the NOX activity assay with fulvene-5, a specific NOX4 inhibitor. Data show that NOX4 activity was significantly increased by I/R, and reduces to baseline with netrin-1 perfusion (p b 0.05, n = 3). G) Mitochondrial swelling assay from I/R-injured hearts with or without netrin-1 perfu- sion. Mitochondrial swelling was measured as an assessment of mitochondrial integrity. Summarized data show that during the monitoring time of 20 min, calcium induced swelling of mitochondria was markedly increased in I/R-injured heart. Perfusion with netrin-1 attenuated this response to baseline (p b =0.001 vs. all others, n = 4). H) Respiratory control ratio, a measure of mitochondrial function, was measured as the ratio of state III (ADP stimulated) verses state IV (oligomycin inhibited) oxygen consumption rate (n = 4, p b 0.05). I) H2O2 as measured with Amplex red (n = 4 each, p b 0.01 vs all) shows I/R significantly increasing H2O2 levels compared with controls, while netrin-1 treatment abolishes this response. J) TTC from I/R-injured hearts after being perfused with netrin-1 or co-perfused with the cGMP inhibitor Rp-8-Br-PET-cGMP (n = 4). The results show that inhibition of cGMP completely eliminated netrin-1's cardioprotective effect. K) Detection of S-nitrosylated proteins from I/R-injured hearts, with or without netrin-1 perfusion. The results show that there were no significant changes in S-nitrosylation of proteins with I/R or perfusion of netrin-1.

Article Snippet: The procedures for this control experiment were: bovine aortic endothelial cells cultured as previously described [29] at passages 4–5 were transfected with pCMV-XL5 human NOX4 plasmid (Origene, Cat#SC310253).

Techniques: Activity Assay, Quantitative Proteomics, Electron Paramagnetic Resonance, Control, Membrane, Western Blot, Expressing, Plasmid Preparation, Inhibition

Journal: Journal of molecular and cellular cardiology

Article Title: Netrin-1 abrogates ischemia/reperfusion-induced cardiac mitochondrial dysfunction via nitric oxide-dependent attenuation of NOX4 activation and recoupling of NOS.

doi: 10.1016/j.yjmcc.2014.07.005

Figure Lengend Snippet: Fig. 2. Nitric oxide (NO) mediates netrin-1 downregulation of NOX4 protein abundance. A) NOX4 protein level in I/R injured hearts that were subjected to netrin-1 with or without NO scavenger PTIO. The reduction in NOX4 protein level in netrin-1 treated hearts was abolished by co-treatment with PTIO (p b 0.01 vs. all others, n = 3). B) NOX4 protein level in freshly isolated adult cardiomyocytes was significantly downregulated by the NO donor MAMANOATE (1 mmol/L) (p = 0.003 vs. control, n = 3).

Article Snippet: The procedures for this control experiment were: bovine aortic endothelial cells cultured as previously described [29] at passages 4–5 were transfected with pCMV-XL5 human NOX4 plasmid (Origene, Cat#SC310253).

Techniques: Quantitative Proteomics, Isolation, Control

Journal: Redox biology

Article Title: NOX4 promotes ferroptosis of astrocytes by oxidative stress-induced lipid peroxidation via the impairment of mitochondrial metabolism in Alzheimer's diseases.

doi: 10.1016/j.redox.2021.101947

Figure Lengend Snippet: Fig. 1. The levels of NOX4 are elevated in impaired astrocytes of the cortex region from patients with Alzheimer’s diseases. (A) Representative immunofluorescence im ages of NOX4 protein expression in cerebral cortex region from patients with AD (AD #1, AD #2, AD #3) or non-AD (normal) showing NOX4 (green) in astrocytes expressing as trocytes marker GFAP (red) around molec ular layer (ML) (n = 3 per group, n = 10 images per individual subject). DAPI-stained nuclei are shown in blue. OS, Outer surface; ML, Molecular layer; EGL, External granular layer. Scale bars, 20 μm. White arrows indicate NOX4 and GFAP positive cells. Symbols, which are expressed by white dotted line, indicate the distinct area among OS, ML, and EGL. (B) Quantification of in tensity for NOX4 positive staining in astro cytes from immunofluorescence images in the cerebral cortex region from patients with AD (AD #1, AD #2, AD #3) or non-AD (normal) (n = 3 per group, n = 10 images per individual subject). Data are mean ± standard deviation (SD). **, p < 0.01 by Student’s two-tailed t-test. (C) Quantifica tion of NOX4 positive astrocytes from immunofluorescence images in the cerebral cortex region from patients with AD (AD #1, AD #2, AD #3) or non-AD (normal) (n = 3 per group, n = 10 images per individual subject). Data are mean ± standard devia tion (SD). **, p < 0.01 by Student’s two- tailed t-test. (For interpretation of the refer ences to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Cells were transduced with pCMV6-AC-GFP constructs against human NOX4 (NM_016931) (RG208007, Origene, Rockville, MD, USA) or pCMV6-ACGFP vector (PS100010, Origene).

Techniques: Immunofluorescence, Expressing, Marker, Staining, Standard Deviation, Two Tailed Test

Journal: Redox biology

Article Title: NOX4 promotes ferroptosis of astrocytes by oxidative stress-induced lipid peroxidation via the impairment of mitochondrial metabolism in Alzheimer's diseases.

doi: 10.1016/j.redox.2021.101947

Figure Lengend Snippet: Fig. 3. The levels of NOX4 are elevated in impaired astrocytes of the cortex region from brain of APP/PS1 mice. (A) Representative immunofluorescence images of NOX4 protein expression in cortex region from brain of APP/PS1 mice (APP/PS1) or wild-type mice (WT) showing NOX4 (green) in astrocytes expressing astrocytes marker GFAP (red) (n = 5 per group, n = 10 images per individual subject). DAPI-stained nuclei are shown in blue. Scale bars, 20 μm. White arrows indicate NOX4 and GFAP positive cells. (B) Quantification of intensity for NOX4 positive staining in astrocytes from immunoflu orescence images in the cortex region from brains of APP/PS1 mice (APP/PS1) or wild-type mice (WT) (n = 5 per group, n = 10 images per individual subject). Data are mean ± standard deviation (SD). **, p < 0.01 by Student’s two-tailed t-test. (C) Quantification of NOX4 positive astrocytes from immunofluorescence images in the cortex region from brains of APP/PS1 mice (APP/PS1) or wild-type mice (WT) (n = 5 per group, n = 10 images per individual subject). Data are mean ± standard deviation (SD). **, p < 0.01 by Student’s two-tailed t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Cells were transduced with pCMV6-AC-GFP constructs against human NOX4 (NM_016931) (RG208007, Origene, Rockville, MD, USA) or pCMV6-ACGFP vector (PS100010, Origene).

Techniques: Immunofluorescence, Expressing, Marker, Staining, Standard Deviation, Two Tailed Test

Journal: Redox biology

Article Title: NOX4 promotes ferroptosis of astrocytes by oxidative stress-induced lipid peroxidation via the impairment of mitochondrial metabolism in Alzheimer's diseases.

doi: 10.1016/j.redox.2021.101947

Figure Lengend Snippet: Fig. 5. The elevation of NOX4 promotes oxidative stress by impairment of mitochondrial metabolism via inhibition of mitochondrial respiration and ATP production in human astrocytes. (A) The levels of oxygen consumption rate (OCR) as the parameter of mitochondrial respiration activity and (B) quantification of OCR levels in control (Control) and NOX4 overexpressing (NOX4) human astrocytes. Data are representative of three independent experiments. Data are mean ± SEM. **p < 0.01; *p < 0.05 using two- tailed Student’s t-test. (C) Representative immunoblot analysis for five mitochondrial ETC protein levels (left) including NDUFB8 for Complex I (C I (NDUFB8)), SDHB for Complex II (C II (SDHB)), UQCRC2 for Complex III (C III (UQCRC2)), MTCO1 for Complex IV (C IV (MTCO1)) and ATP5F1A for Complex V (C V (ATP5F1A)) in control (Control) and NOX4 overexpressing (NOX4) human astrocytes. Quantification for protein levels of C I (NDUFB8, C II (SDHB), C III (UQCRC2), C IV (MTCO1) and C V (ATP5F1A) (right) in control (Control) and NOX4 overexpressing (NOX4) human astrocytes. For immunoblots, β-actin was used as a loading control. Data are representatives of three independent experiments. Data are mean ± standard deviation (SD). **p < 0.01; *p < 0.05 using two-tailed Student’s t-test. (D) Quantification of mitochondrial ATP production rate in control (Control) and NOX4 overexpressing (NOX4) human astrocytes. Data are mean ± SD. **p < 0.01 using two-tailed Student’s t-test. (E) Quantification of mtROS levels using MitoSOX staining in control (Control) and NOX4 overexpressing (NOX4) human astrocytes. Data are mean ± SD. *p < 0.05 using two-tailed Student’s t-test. (F) Representative immunofluorescence images of mitochondrial morphology for mitochondria fragmentation by Tomm20 staining in control (Control) and NOX4 overexpressing (NOX4) human astrocytes showing Tomm20 (green) (n = 10 per group). DAPI- stained nuclei are shown in blue. The fragmentation of mitochondria is indicated (white arrows). Scale bars, 20 μm. Magnified views of the selected regions (upper right); scale bars, 5 μm. (G) Quantification of cells with mitochondrial fragmentation from immunofluorescence images of mitochondrial morphology in control (Control) and NOX4 overexpressing (NOX4) human astrocytes (n = 10 per group). (The percent of morphological dead cells in a total of 100 cells of 10 individual images per group was calculated). Symbols expressed by white dotted line indicate the shape of cells. Data are mean ± SD. **, p < 0.01 by Student’s two-tailed t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Cells were transduced with pCMV6-AC-GFP constructs against human NOX4 (NM_016931) (RG208007, Origene, Rockville, MD, USA) or pCMV6-ACGFP vector (PS100010, Origene).

Techniques: Inhibition, Activity Assay, Control, Two Tailed Test, Western Blot, Standard Deviation, Staining, Immunofluorescence

Journal: Redox biology

Article Title: NOX4 promotes ferroptosis of astrocytes by oxidative stress-induced lipid peroxidation via the impairment of mitochondrial metabolism in Alzheimer's diseases.

doi: 10.1016/j.redox.2021.101947

Figure Lengend Snippet: Fig. 6. NOX4-induced mitochondrial metabolic impairment induces oxidative stress by inhibition of cellular antioxidant process in human astrocytes. (A-C) Quantification of (A) reduced GSH levels, (B) ratio of GSH2/GSSG, and (C) GSSG levels in control (Control) and NOX4 overexpressing (NOX4) human astrocytes (n = 10 per group). Data are mean ± SD. *, p < 0.01 by Student’s two-tailed t-test. (D) Representative immunoblot analysis for nuclear and cytosolic NRF2 in control (Control) and NOX4 overexpressing (NOX4) human astrocytes. For immunoblots, Histone H3 (nuclear) and β-actin (cytosolic) was used as a loading control. Data are representative of three independent experiments. Data are mean ± SD. **, p < 0.01; *, p < 0.05 using the two-tailed Student’s t-test. (E–F) Quantification of (E) HO-1 protein levels, (F) HO-1 activity, (G) GCLC protein levels and (F) GCL activity in control (Control) and NOX4 overexpressing (NOX4) human astrocytes (n = 10 per group). Data are mean ± SD. *, p < 0.01 by Student’s two-tailed t-test.

Article Snippet: Cells were transduced with pCMV6-AC-GFP constructs against human NOX4 (NM_016931) (RG208007, Origene, Rockville, MD, USA) or pCMV6-ACGFP vector (PS100010, Origene).

Techniques: Inhibition, Control, Two Tailed Test, Western Blot, Activity Assay

Journal: Redox biology

Article Title: NOX4 promotes ferroptosis of astrocytes by oxidative stress-induced lipid peroxidation via the impairment of mitochondrial metabolism in Alzheimer's diseases.

doi: 10.1016/j.redox.2021.101947

Figure Lengend Snippet: Fig. 7. NOX4 promotes ferroptosis by oxidative stress-induced lipid peroxidation in human astrocytes. (A) Representative immunofluorescence images of 4-HNE expression in control (Control) and NOX4 overexpressing (NOX4) human astrocytes showing 4-HNE (red) (n = 10 per group). DAPI-stained nuclei are shown in blue. The shape of cells showed shrinkage and lipid peroxidation-derived droplets in the plasma membrane were indicated (white arrows). Symbols expressed by white dotted line indicate the shape of cells. Scale bars, 20 μm. (B) Quantification of 4-HNE positive astrocytes from immunofluorescence images in control (Control) and NOX4 overexpressing (NOX4) human astrocytes (n = 10 per group) (The percent of morphological dead cells in a total of 100 cells of 10 individual images per group was calculated). Data are mean ± standard deviation (SD). **, p < 0.01 by Student’s two-tailed t-test. (C) Representative immunoblot analysis for 4-HNE and MDA protein levels (left) and quantification for 4-HNE and MDA protein levels in control (Control) and NOX4 overexpressing (NOX4) human astrocytes. For immunoblots, β-actin was used as a loading control. Data are representative of three independent experiments. Data are mean ± SD. *p < 0.05 using the two-tailed Student’s t-test. (D) Quantification of iron levels in control (Control) and NOX4 overexpressing (NOX4) human astrocytes (n = 10 per group). Data are mean ± standard deviation (SD). *, p < 0.05 by Student’s two-tailed t-test. (E) Representative 3D images of control (Control) and NOX4 overexpressing (NOX4) human astrocytes (n = 10 images per group). The morphological features of cytotoxicity were indicated (white arrows). Scale bars, 20 μm. (F) Quantification of the morphological dead cells in control (Control) and NOX4 overexpressing (NOX4) human astrocytes (n = 10 per group) (The percent of morphological dead cells in total 100 cells in 10 individual images per group). Data are mean ± SD. **, p < 0.01 using the two-tailed Student’s t-test. (G) Cytotoxicity assay in control (Control) and NOX4 overexpressing (NOX4) human astrocytes was determined by lactate dehydrogenase (LDH) levels. Data are representatives of three independent experiments. Each experiment was done in triplicate. Data are mean ± SD. **, p < 0.01 using two-tailed Student’s t-test. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Cells were transduced with pCMV6-AC-GFP constructs against human NOX4 (NM_016931) (RG208007, Origene, Rockville, MD, USA) or pCMV6-ACGFP vector (PS100010, Origene).

Techniques: Immunofluorescence, Expressing, Control, Staining, Derivative Assay, Clinical Proteomics, Membrane, Standard Deviation, Two Tailed Test, Western Blot, Cytotoxicity Assay