Journal: International Journal of Molecular Sciences

Article Title: PM2.5 Exposure Triggers Hypothalamic Oxidative and ER Stress Leading to Depressive-like Behaviors in Rats

doi: 10.3390/ijms252413527

Figure Lengend Snippet: PM2.5 nasal administration increases protein and mRNA levels of NOX4 in the hypothalamus but not in the hippocampus. ( A , D ) Western blot analysis of NOX4 protein levels in the hypothalamus ( A ) and hippocampus ( D ) following vehicle or PM2.5 treatment. β-actin served as a loading control. ( B , E ) Quantification of NOX4 protein levels from panels A and D, respectively. ( C , F ) Real-time qPCR for NOX4 mRNA level in the ( C ) hypothalamus and ( F ) hippocampus following vehicle or PM2.5 treatment. Data are presented as mean ± SEM, with comparisons made using Student’s t -test in panels ( B , D ). ns., not significant. * p ≤ 0.05 vs. Vehicle, n = 4.

Article Snippet: The primary antibodies used for immunoblotting were: GAPDH (diluted 1:2500, sc-365062) and TH (diluted 1:1000, sc-25269) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); VAChT (diluted 1:1000, #75-020) from NeuroMab Facility (UC Davis/NIH, CA, USA); β-actin (diluted 1:2500, ab6276) and β-III-tubulin (#ab7751, diluted 1:1000) from Abcam (Cambridge, UK); NOX4 (diluted 1:1000, NB110-58849) from Novus Biologicals (Littleton, CO, USA).

Techniques: Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: PM2.5 Exposure Triggers Hypothalamic Oxidative and ER Stress Leading to Depressive-like Behaviors in Rats

doi: 10.3390/ijms252413527

Figure Lengend Snippet: PM2.5 induces ER stress and mitochondrial ROS production, reducing neuronal markers in Neuro-2A cells. ( A ) q-PCR analysis of ER stress markers ATF4 and CHOP in Neuro-2A cells treated with low (1 μg/mL) and high (100 μg/mL) doses of PM2.5. ( B ) Western blot analysis of NOX4 protein levels in Neuro-2A cells treated with vehicle, 1 μg/mL, and 100 μg/mL of PM2.5, with β-actin as a loading control. ( C , D ) Mitochondrial ROS production in Neuro-2A cells treated with PM2.5 (100 μg/mL), showing a significant increase in ROS levels compared to vehicle control. ( E , F ) PM2.5-induced mitochondrial ROS increase was significantly reduced by NOX inhibitors GKT137831 and Apocynin. ( G ) Western blot analysis of β-III-tubulin and VAChT protein levels in Neuro-2A cells treated with increasing concentrations of PM2.5 (1, 3, 10, 30, and 100 μg/mL), with β-actin as a loading control. ( H , I ) Quantifying β-III-tubulin ( H ) and VAChT ( I ) protein levels from panel G, respectively. Scale bars, 5 μm. Bar graphs are expressed as mean ± SEM and analyzed using one-way ANOVA ( A , D , F , H , I ). * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.001 vs. Vehicle. # p ≤ 0.05 vs. PM2.5.

Article Snippet: The primary antibodies used for immunoblotting were: GAPDH (diluted 1:2500, sc-365062) and TH (diluted 1:1000, sc-25269) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); VAChT (diluted 1:1000, #75-020) from NeuroMab Facility (UC Davis/NIH, CA, USA); β-actin (diluted 1:2500, ab6276) and β-III-tubulin (#ab7751, diluted 1:1000) from Abcam (Cambridge, UK); NOX4 (diluted 1:1000, NB110-58849) from Novus Biologicals (Littleton, CO, USA).

Techniques: Western Blot, Control

Journal: Antioxidants

Article Title: Specific NOX4 Inhibition Preserves Mitochondrial Function and Dampens Kidney Dysfunction Following Ischemia–Reperfusion-Induced Kidney Injury

doi: 10.3390/antiox13040489

Figure Lengend Snippet: ( A ) Glomerular filtration rate (GFR) (N = 6/group) and ( B ) blood urea nitrogen (N = 6/group) was evaluated in mice treated with NOX4 inhibitor (NOX4i) during the first 24 h and during the whole reperfusion period of 3 days of reperfusion following 20 min bilateral ischemia (AKI). Venous injection of FITC-inulin was performed followed by sequential sampling of plasma. Plasma clearance of FITC-inulin was evaluated spectrofluorometrically. BUNs were measured by using a commercial colorimetric detection kit. One-Way ANOVA was used for statistical analysis. * for p ≤ 0.05, and **** for p ≤ 0.0001.

Article Snippet: The primary antibodies used were mouse anti-OXPHOS antibody cocktail (Thermofisher Scientific, Waltham, MA, USA, 45-8099), rabbit anti-NOX4 antibody (Novus Biologicals, Shanghai, China, NB110-58849SS), rabbit anti phospo-Nrf2 (Ser40) (Thermofisher, Thermofisher Scientific, Waltham, MA, USA, PA5-67520), mouse anti- alpha tubulin ( Novus Biologicals, NB100-690 ).

Techniques: Filtration, Injection, Sampling, Clinical Proteomics

Journal: Antioxidants

Article Title: Specific NOX4 Inhibition Preserves Mitochondrial Function and Dampens Kidney Dysfunction Following Ischemia–Reperfusion-Induced Kidney Injury

doi: 10.3390/antiox13040489

Figure Lengend Snippet: Histopathological evaluation of kidneys in the cortico–medullary junction (S3 segment area) in animals exposed to bilateral ischemia (20 min), treated with NOX4 inhibitor (NOX4i) during the first 24 h or 76 h of the 3-day reperfusion phase (N = 6/group). ( A ) Representative images of paraformaldehyde-fixed kidney slices stained with hematoxylin–eosin (HE) and periodic acid-Schiff (PAS) analyzed under light microscopy. (( A ), A–B ) Control group (SHAM) with normal tubular appearance, preserved cell morphology, and presence of an intact brush border positively highlighted by PAS (black arrow), in the region close to the renal medulla (M). (( A ), C–D ) Samples from the experimental group exposed to ischemia–reperfusion-induced kidney injury (AKI) showing extensive dilation of the S3 segment area tubules, cast formation, and sloughing of tubular epithelial cells or loss of the brush border (black arrowhead). (( A ), E–F ) Samples from the group exposed to AKI treated with specific NOX4i during the first 24 h of reperfusion showing a non-significant reduction in tubular damage in the S3 region close to the renal medulla (M), the majority of tubules with a normal diameter and cellular integrity (*), and other tubules presenting cellular debris in the lumen (white arrow) and the formation of hyaline casts (white arrowhead). (( A ), G–H ) Samples from the group with specific NOX4i for 76 h, with a significant reduction in tubular alterations, observed by the reduction in tubular dilation and greater integrity of cells and the brush border (#), with some tubules showing atrophy and the formation of hyaline casts (red arrowhead). ( B ) Statistical analysis of tubular injury score (Kruskal–Wallis test). Score 0: no tubular injury; Score 1: <10% of tubules injured; Score 2: 10–25% of tubules injured; Score 3: 25–50% of tubules injured; Score 4: 50–74% of tubules injured; Score 5: >75% of tubules injured. * for p ≤ 0.05, and **** for p ≤ 0.0001. Non-significant variations are indicated as ns.

Article Snippet: The primary antibodies used were mouse anti-OXPHOS antibody cocktail (Thermofisher Scientific, Waltham, MA, USA, 45-8099), rabbit anti-NOX4 antibody (Novus Biologicals, Shanghai, China, NB110-58849SS), rabbit anti phospo-Nrf2 (Ser40) (Thermofisher, Thermofisher Scientific, Waltham, MA, USA, PA5-67520), mouse anti- alpha tubulin ( Novus Biologicals, NB100-690 ).

Techniques: Staining, Light Microscopy, Control

Journal: Antioxidants

Article Title: Specific NOX4 Inhibition Preserves Mitochondrial Function and Dampens Kidney Dysfunction Following Ischemia–Reperfusion-Induced Kidney Injury

doi: 10.3390/antiox13040489

Figure Lengend Snippet: A TUNEL assay was performed in kidney slices to evaluate apoptosis in the kidney S3 segment following acute kidney injury (AKI) (N = 6/group) 20 min bilateral ischemia followed by 3 days of reperfusion) combined with NOX4 inhibition (NOX4i) during either the first 24 h or the whole 76 h of reperfusion. ( A ) Representative TUNEL-stained kidney slices, where brown areas represent apoptotic tissue. ( B ) The percentage of TUNEL-positive area on the 3rd day of reperfusion was estimated by using the particle analysis command in the ImageJ software. One-Way ANOVA was used for statistical analysis. * for p ≤ 0.05. Non-significant variations are indicated as ns.

Article Snippet: The primary antibodies used were mouse anti-OXPHOS antibody cocktail (Thermofisher Scientific, Waltham, MA, USA, 45-8099), rabbit anti-NOX4 antibody (Novus Biologicals, Shanghai, China, NB110-58849SS), rabbit anti phospo-Nrf2 (Ser40) (Thermofisher, Thermofisher Scientific, Waltham, MA, USA, PA5-67520), mouse anti- alpha tubulin ( Novus Biologicals, NB100-690 ).

Techniques: TUNEL Assay, Inhibition, Staining, Particle Size Analysis, Software

Journal: Antioxidants

Article Title: Specific NOX4 Inhibition Preserves Mitochondrial Function and Dampens Kidney Dysfunction Following Ischemia–Reperfusion-Induced Kidney Injury

doi: 10.3390/antiox13040489

Figure Lengend Snippet: The degree of mitochondrial coupling and hydrogen peroxide production were evaluated on the 3rd day of reperfusion in isolated mitochondria from kidneys in mice following induced acute kidney injury (AKI) (20 min bilateral ischemia) combined with NOX4 inhibition (NOX4i). ( A ) The mitochondrial respiratory control ratio (RCR) defined as maximal CI-dependent state 3 respiration related to state 2 respiration in absence of adenylates was evaluated by high-resolution respirometry (Sham N = 7, AKI 3d and AKI 3d + NOX4i 76 h N = 6, NOX4i 24 h N = 5 and naïve + NOX4i 76 h N = 3) ( B ). Mitochondrial hydrogen peroxide (H 2 O 2 ) production was measured spectrofluorometrically during leak respiration by using the amplex red system (N = 6/group, except for NOX4i 24 h N = 5). Note that NOX4i was washed away during the mitochondrial isolation protocol during these experiments. * for p ≤ 0.05, ** for p ≤ 0.01, and *** for p ≤ 0.001. Non-significant variations are indicated as ns.

Article Snippet: The primary antibodies used were mouse anti-OXPHOS antibody cocktail (Thermofisher Scientific, Waltham, MA, USA, 45-8099), rabbit anti-NOX4 antibody (Novus Biologicals, Shanghai, China, NB110-58849SS), rabbit anti phospo-Nrf2 (Ser40) (Thermofisher, Thermofisher Scientific, Waltham, MA, USA, PA5-67520), mouse anti- alpha tubulin ( Novus Biologicals, NB100-690 ).

Techniques: Isolation, Inhibition, Control

Journal: Antioxidants

Article Title: Specific NOX4 Inhibition Preserves Mitochondrial Function and Dampens Kidney Dysfunction Following Ischemia–Reperfusion-Induced Kidney Injury

doi: 10.3390/antiox13040489

Figure Lengend Snippet: Kidney mitochondrial respiratory complex activities were evaluated by high-resolution respirometry on the 3rd day of reperfusion in isolated mitochondria from mice following induced acute kidney injury (AKI) (20 min bilateral ischemia) combined with NOX4 inhibition (NOX4i) during the first 24 h or during the whole reperfusion period (Sham N = 7, AKI 3d and AKI 3d + NOX4i 76h N = 6, NOX4i 24 h N = 5 and naïve + NOX4i 76 h N = 3). A control group of naïve animals were administered NOX4i to evaluate the effect on mitochondrial function. State 3 respiration in presence of adenylates was determined for each mitochondrial complex activity, respectively. ( A – D ) CI-CIV activity was normalized to mitochondrial protein. * for p ≤ 0.05, ** for p ≤ 0.01, *** for p ≤ 0.001, and **** for p ≤ 0.0001.

Article Snippet: The primary antibodies used were mouse anti-OXPHOS antibody cocktail (Thermofisher Scientific, Waltham, MA, USA, 45-8099), rabbit anti-NOX4 antibody (Novus Biologicals, Shanghai, China, NB110-58849SS), rabbit anti phospo-Nrf2 (Ser40) (Thermofisher, Thermofisher Scientific, Waltham, MA, USA, PA5-67520), mouse anti- alpha tubulin ( Novus Biologicals, NB100-690 ).

Techniques: Isolation, Inhibition, Control, Activity Assay

Journal: Antioxidants

Article Title: Specific NOX4 Inhibition Preserves Mitochondrial Function and Dampens Kidney Dysfunction Following Ischemia–Reperfusion-Induced Kidney Injury

doi: 10.3390/antiox13040489

Figure Lengend Snippet: Kidney tissue citrate synthase activity was measured in mice after the induction of acute kidney injury (AKI) by 20 min ischemia followed by treatment with NOX4 inhibitor (NOX4i) during the reperfusion phase (N = 6/group except for AKI 3d + NOX4i 76 h, N = 5). Citrate synthase activity was measured by using a commercial colorimetric kit. One-Way ANOVA was used for statistical analysis. Non-significant variations were indicated as ns.

Article Snippet: The primary antibodies used were mouse anti-OXPHOS antibody cocktail (Thermofisher Scientific, Waltham, MA, USA, 45-8099), rabbit anti-NOX4 antibody (Novus Biologicals, Shanghai, China, NB110-58849SS), rabbit anti phospo-Nrf2 (Ser40) (Thermofisher, Thermofisher Scientific, Waltham, MA, USA, PA5-67520), mouse anti- alpha tubulin ( Novus Biologicals, NB100-690 ).

Techniques: Activity Assay

Journal: Antioxidants

Article Title: Specific NOX4 Inhibition Preserves Mitochondrial Function and Dampens Kidney Dysfunction Following Ischemia–Reperfusion-Induced Kidney Injury

doi: 10.3390/antiox13040489

Figure Lengend Snippet: Immunoblotting was performed to evaluate the protein levels of the mitochondrial respiratory complexes in kidneys on the 3rd day of reperfusion (AKI) in animals treated with the NOX4 inhibitor (NOX4i) (N = 4/group). ( A ) Protein expression levels using a cocktail of antibodies targeting mitochondrial complexes. ( B ) Protein staining of the membrane used for normalization. ( C – F ) Statistical analysis of the protein levels of CI, CII, CIII, and CV (One-Way ANOVA).

Article Snippet: The primary antibodies used were mouse anti-OXPHOS antibody cocktail (Thermofisher Scientific, Waltham, MA, USA, 45-8099), rabbit anti-NOX4 antibody (Novus Biologicals, Shanghai, China, NB110-58849SS), rabbit anti phospo-Nrf2 (Ser40) (Thermofisher, Thermofisher Scientific, Waltham, MA, USA, PA5-67520), mouse anti- alpha tubulin ( Novus Biologicals, NB100-690 ).

Techniques: Western Blot, Expressing, Staining, Membrane

Journal: Antioxidants

Article Title: Specific NOX4 Inhibition Preserves Mitochondrial Function and Dampens Kidney Dysfunction Following Ischemia–Reperfusion-Induced Kidney Injury

doi: 10.3390/antiox13040489

Figure Lengend Snippet: Immunoblotting was performed to evaluate protein levels of Ser40 phosphorylated Nrf2 in kidneys on the 3rd day of reperfusion (AKI) in animals treated with NOX4 inhibitor (NOX4i) (Sham N = 5, AKI 3d N = 6, AKI 3d + NOX4i 24 h N = 5, AKI3d + NOX4i N = 6). ( A ) Protein expression using antibodies targeting phosphorylated Nrf2 (Ser40), Nrf2, and membranes stained for protein used for normalization. Corrections between blots were made using a control sample denoted * . ( B ) Statistical analysis of the levels of phosphorylated Nrf2 related to Nrf2 and ( C ) total protein. ( D ) Nrf2 normalized to total protein. One-Way ANOVA was used for statistical analysis. * for p ≤ 0.05, ** for p ≤ 0.01, and *** for p ≤ 0.001. Non-significant variations are indicated as ns.

Article Snippet: The primary antibodies used were mouse anti-OXPHOS antibody cocktail (Thermofisher Scientific, Waltham, MA, USA, 45-8099), rabbit anti-NOX4 antibody (Novus Biologicals, Shanghai, China, NB110-58849SS), rabbit anti phospo-Nrf2 (Ser40) (Thermofisher, Thermofisher Scientific, Waltham, MA, USA, PA5-67520), mouse anti- alpha tubulin ( Novus Biologicals, NB100-690 ).

Techniques: Western Blot, Expressing, Staining, Control

Journal: Antioxidants

Article Title: Specific NOX4 Inhibition Preserves Mitochondrial Function and Dampens Kidney Dysfunction Following Ischemia–Reperfusion-Induced Kidney Injury

doi: 10.3390/antiox13040489

Figure Lengend Snippet: ( A ) Immunoblotting by targeting NOX4 was performed on HK-2 cells exposed to hypoxia (2% O 2 ) for 3 h followed by reoxygenation for 24 h (H/R). ( B ) Statistical analysis of the NOX4 protein levels normalized to α-tubulin by using Student’s t -test. * for p ≤ 0.05.

Article Snippet: The primary antibodies used were mouse anti-OXPHOS antibody cocktail (Thermofisher Scientific, Waltham, MA, USA, 45-8099), rabbit anti-NOX4 antibody (Novus Biologicals, Shanghai, China, NB110-58849SS), rabbit anti phospo-Nrf2 (Ser40) (Thermofisher, Thermofisher Scientific, Waltham, MA, USA, PA5-67520), mouse anti- alpha tubulin ( Novus Biologicals, NB100-690 ).

Techniques: Western Blot