Journal: The American Journal of Pathology

Article Title: 7-Ketocholesterol Induces Autophagy in Vascular Smooth Muscle Cells through Nox4 and Atg4B

doi: 10.1016/j.ajpath.2013.04.028

Figure Lengend Snippet: 7-KC–induced autophagy is mediated by Nox4-derived hydrogen peroxide. A: HASMCs were incubated with 20 μmol/L 7-KC for 16 hours. The mRNA levels of Nox1, Nox4, and Nox5 were measured by using real-time PCR (n = 4). ∗P < 0.05 versus control (Con). B and C: Protein levels of Nox1, Nox4, and Nox5 were determined by using Western blot analysis and quantified by using densitometry (n = 4). ∗P < 0.05 versus Con. D: HASMCs were treated with 7-KC (20 μmol/L) for indicated times. Nox4 protein levels in cell lysates were analyzed by using Western blot analysis (upper panel) and quantified by using densitometry (lower panel) (n = 4). P < 0.05 versus Con. E: HASMCs were incubated with 20 μmol/L 7-KC for the indicated times, and hydrogen peroxide was detected by using a hydrogen peroxide cell-based assay kit (n = 5). ∗P < 0.05 versus Con. F: HASMCs were infected with adenovirus encoding GFP or catalase (Cata) for 48 hours and then incubated with 7-KC (20 μmol/L) for 16 hours. Protein levels of LC3-II and catalase were detected by using Western blot analysis (upper panel) and quantified by using densitometry (lower panel) (n = 5). ∗P < 0.05 versus GFP or Con, †P < 0.05 versus GFP/7-KC.

Article Snippet: Reagents We used the following antibodies: antibodies against Beclin1, LC3B, Atg4B, and phosphorylated eukaryotic initiation factor-2 α (P-eIF2α) (Cell Signaling Inc., Beverly, MA), antibodies against Atg4A and 78 kDa glucose-regulated protein (GRP78) (Abcam, Cambridge, MA), antibodies against catalase and reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase (Nox) subunits (Nox4, catalog number sc-55142; Nox5, catalog number sc-67006) (Santa Cruz Biotechnology, Santa Cruz, CA), antibody against Nox1 (catalog number NBP1-69573; Novus Biologicals, Littleton, CO), and the antibody against activating transcription factor 6 (Imgenex, San Diego, CA).

Techniques: Derivative Assay, Incubation, Real-time Polymerase Chain Reaction, Control, Western Blot, Cell Based Assay, Infection

Journal: The American Journal of Pathology

Article Title: 7-Ketocholesterol Induces Autophagy in Vascular Smooth Muscle Cells through Nox4 and Atg4B

doi: 10.1016/j.ajpath.2013.04.028

Figure Lengend Snippet: The 7-KC–stimulated autophagy is mediated by inhibition of Atg4B. HASMCs were transfected with control or Beclin1 siRNA for 48 hours and then treated with 7-KC (20 μmol/L) for 16 hours. Expression of Beclin1 and LC3-II were measured by using Western blot analysis (A, upper panel) and quantified by using densitometry (A, lower panel) (n = 5). ∗P < 0.05 versus C-siRNA. HASMCs were treated with 7-KC (20 μmol/L) for 16 hours and the expression of Atg4A and Atg4B was measured by using Western blot analysis (B, upper panel). Atg4B activity in cell lysates was assayed as described in Materials and Methods (B, lower panel) (n = 6). ∗P < 0.05 versus control (Con). HASMCs were transfected with control or Nox4 siRNA for 48 hours, and protein levels of Nox1, Nox4, and Nox5 were assessed by using Western blot analysis (C) and quantified by using densitometry (D) (n = 3). ∗P < 0.05 versus Con. E: HASMCs were transfected with control or Nox4 siRNA for 48 hours and were then treated with 20 μmol/L 7-KC for 16 hours. Hydrogen peroxide was measured by using a hydrogen peroxide cell-based assay kit (n = 4). ∗P < 0.05 versus C-siRNA, †P < 0.05 versus C-siRNA/7-KC. F: Atg4B activity in cell lysates was assayed as described in Materials and Methods (n = 5). ∗P < 0.05 versus C-siRNA, †P < 0.05 versus C-siRNA/7-KC. G: Expression of LC3-II and Nox4 was examined by using Western blot analysis (n = 4). ∗P < 0.05 versus C-siRNA; †P < 0.05 versus C-siRNA/7-KC.

Article Snippet: Reagents We used the following antibodies: antibodies against Beclin1, LC3B, Atg4B, and phosphorylated eukaryotic initiation factor-2 α (P-eIF2α) (Cell Signaling Inc., Beverly, MA), antibodies against Atg4A and 78 kDa glucose-regulated protein (GRP78) (Abcam, Cambridge, MA), antibodies against catalase and reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase (Nox) subunits (Nox4, catalog number sc-55142; Nox5, catalog number sc-67006) (Santa Cruz Biotechnology, Santa Cruz, CA), antibody against Nox1 (catalog number NBP1-69573; Novus Biologicals, Littleton, CO), and the antibody against activating transcription factor 6 (Imgenex, San Diego, CA).

Techniques: Inhibition, Transfection, Control, Expressing, Western Blot, Activity Assay, Cell Based Assay

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: Mechanobiology in Medicine

Article Title: Overstretch causes lipid accumulation in vascular smooth muscle cells dependent on NADPH oxidase 1

doi: 10.1016/j.mbm.2025.100129

Figure Lengend Snippet: Roles of NOX1 and ROS in the stretch-induced lipid accumulation in VSMCs. (A) NOX1 transcription and protein expression; (B) ROS level indicated by DCFH, DHE, Deep Red and MitoSOX; (C) Stretch-induced lipid accumulation under different antioxidant treatment, ML-090 (10 ​μM), Apocynin (300 ​μM), NAC (1 ​mM), catalase (500 IU), SOD (100 IU). The data are shown as mean ​± ​SD (n ​= ​3), ∗ p ​< ​0.05, ∗∗ p ​< ​0.01, ∗∗∗ p ​< ​0.001, ∗∗∗∗ p ​< ​0.0001.

Article Snippet: Antibodies against NOX1 (1:2000, CSB- PA443876 ), NOX3 (1:2000, CSB-PA015960ESR2HU), and DUOX2 (1:2000, CSB-PA007229ESR1HU) were obtained from Cusabio (Wuhan, China).

Techniques: Expressing

Journal: Mechanobiology in Medicine

Article Title: Overstretch causes lipid accumulation in vascular smooth muscle cells dependent on NADPH oxidase 1

doi: 10.1016/j.mbm.2025.100129

Figure Lengend Snippet: NOX1 knockdown reduces the stretch-induced lipid accumulation and ROS level in VSMCs. (A) Verification of NOX1 knockdown; (B and C) Lipid accumulation and lipid oxidation indicated by ORO and BODIPY, respectively; (D and E) ROS level indicated by DCFH and DHE. The data are shown as mean ​± ​SD (n ​= ​3), ∗p ​< ​0.05, ∗∗p ​< ​0.01, ∗∗∗p ​< ​0.001, ∗∗∗∗p ​< ​0.0001.

Article Snippet: Antibodies against NOX1 (1:2000, CSB- PA443876 ), NOX3 (1:2000, CSB-PA015960ESR2HU), and DUOX2 (1:2000, CSB-PA007229ESR1HU) were obtained from Cusabio (Wuhan, China).

Techniques: Knockdown

Journal: Mechanobiology in Medicine

Article Title: Overstretch causes lipid accumulation in vascular smooth muscle cells dependent on NADPH oxidase 1

doi: 10.1016/j.mbm.2025.100129

Figure Lengend Snippet: Stretch-induced lipid accumulation depends on the JAK/STAT signaling pathway. (A and B) 15 ​% stretch promotes the STAT1/3 phosphorylation and the expression of IL-6 and IFN-γ. (C–E) Inhibition of STAT3 phosphorylation and NOX1 expression, lipid oxidation, and lipid accumulation under 50 ​μM stattic treatment. The data are shown as mean ​± ​SD (n ​= ​3), ∗ p ​< ​0.05, ∗∗ p ​< ​0.01, ∗∗∗ p ​< ​0.001, ∗∗∗∗ p ​< ​0.0001.

Article Snippet: Antibodies against NOX1 (1:2000, CSB- PA443876 ), NOX3 (1:2000, CSB-PA015960ESR2HU), and DUOX2 (1:2000, CSB-PA007229ESR1HU) were obtained from Cusabio (Wuhan, China).

Techniques: Phospho-proteomics, Expressing, Inhibition

Journal: Metabolic Brain Disease

Article Title: Studying neuroprotective effect of Atorvastatin as a small molecule drug on high glucose-induced neurotoxicity in undifferentiated PC12 cells: role of NADPH oxidase

doi: 10.1007/s11011-016-9883-1

Figure Lengend Snippet: RT-PCR analysis to determine Atorvastatin effect on gene expression of NOX1 ( a ), NOX2 ( b ) , NOX3 ( c ), and NOX4 ( d ) in PC12 cells. Cells were pre-treated with Atorvastatin (1 μm) for 96 h and then exposed to HG (140 mM) for 24 h. Apocynin (10 μM) was used as the Atorvastatin control and added 45 min before HG treatment. The densities of NOX1 ( a ), NOX2 ( b ) , NOX3 ( c ), and NOX4 ( d ) ratio to β-actin were determined. Results are reported as mean ± SEM ( n = 3). *** P < 0.001, vs control and ††† P < 0.001, vs HG

Article Snippet: Antibodies against NOX2, 4 were obtained from Abcam (Cambridge, UK), NOX1 from Aviva system biology (San Diego, Ca, USA) and NOX3 from Sigma (Sigma Aldrich, St Louis, MO, USA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Gene Expression, Control

Journal: Metabolic Brain Disease

Article Title: Studying neuroprotective effect of Atorvastatin as a small molecule drug on high glucose-induced neurotoxicity in undifferentiated PC12 cells: role of NADPH oxidase

doi: 10.1007/s11011-016-9883-1

Figure Lengend Snippet: Western blotting analysis to determine Atorvastatin effect on protein levels of NOX1 ( a ), NOX2 ( b ) , NOX3 ( c ) , and NOX4 ( d ) in PC12 cells. Cells were pre-treated with Atorvastatin (1 μM) for 96 h and then exposed to HG (140 mM) for 24 h. Apocynin (1 μM) was used as the Atorvastatin control and added 45 min before HG treatment. The densities of NOX1 ( a ), NOX2 ( b ) , NOX 3 ( c ), and NOX4 ( d ) ratio to β-actin were determined. Results are reported as mean ± SEM (n = 3). * P < 0.05, ** P < 0.01, and *** P < 0.001, vs control, † P < 0.05, †† P < 0.01, and ††† P < 0.001, vs HG

Article Snippet: Antibodies against NOX2, 4 were obtained from Abcam (Cambridge, UK), NOX1 from Aviva system biology (San Diego, Ca, USA) and NOX3 from Sigma (Sigma Aldrich, St Louis, MO, USA).

Techniques: Western Blot, Control

Journal: Scientific reports

Article Title: Reactive Oxygen Species Mediate the Suppression of Arterial Smooth Muscle T-type Ca 2+ Channels by Angiotensin II.

doi: 10.1038/s41598-018-21899-5

Figure Lengend Snippet: Figure 6. Proximity ligation assay of CaV3.2 and Nox1 in cerebral arterial smooth muscle cells. Representative images revealing the close proximity (<40 nM) of CaV3.2 with Nox1 as demonstrated by the presence of red fluorescent products; nuclei were labelled with DAPI (blue). A similar localization pattern between Nox1 and other vascular T-type channels (CaV3.1) could not be detected. Assay controls were performed with one or both primary antibodies removed. Each experiment was performed on cells for 4 different animals; photomicrographs are representative 10–20 smooth muscle cells per group.

Article Snippet: Primary antibodies against CaV3.2, CaV3.1 and Nox1 were purchased from Novus Biologicals.

Techniques: Proximity Ligation Assay