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

Journal: Endocrine-Related Cancer

Article Title: Intracellular expression of reactive oxygen species-generating NADPH oxidase NOX4 in normal and cancer thyroid tissues

doi: 10.1677/erc-09-0175

Figure Lengend Snippet: Figure 1 Expression of NOX in normal human thyroid tissues and in cultured human thyrocytes, as determined by RT-PCR. Lane M, DNA size markers; lane 1, NOX1; lane 2, NOX2; lane 3, NOX3; lane 4, NOX4 and lane 5, NOX5. Expression of thyroid-specific gene transcripts in human thyrocytes is also reported as internal controls.

Article Snippet: Antibody directed against the human NOX4 primary antibody was purchased from Novus Biologicals (Littleton, CO 80160, USA), and its specificity was tested with crude extracts prepared from HEK293 cells transiently transfected with the vector pcDNA3.1 containing or not human NOX4.

Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction

Journal: Endocrine-Related Cancer

Article Title: Intracellular expression of reactive oxygen species-generating NADPH oxidase NOX4 in normal and cancer thyroid tissues

doi: 10.1677/erc-09-0175

Figure Lengend Snippet: Figure 2 Effect of TSH on NOX4 and p22phox expression in human thyrocytes. Primary human thyrocytes were maintained in 5H medium (without TSH) for 72 h and then incubated with 1 mU/ml TSH for the indicated periods. (A) Time-dependent stimulation of NOX4 and p22phox transcription by TSH. mRNAs quantified by real-time RT-PCR were investigated in triplicates and expressed as meansGS.E.M. of one representative experiment from three independent cultures. Expression values (y-axis) are displayed on based 2 logarithmic scale. (B) Time-dependent stimulation of NOX4 and p22phox protein expressions by TSH. NOX4 is recognized as a single band at 75 kDa by western blot analysis in human thyrocytes expressing the thyroperoxidase (TPO). Crude extracts of human thyrocytes were analyzed by western blotting. (C) Effect of TSH on NOX4-dependent ROS levels in human thyrocytes. ROS levels measured as DCF fluorescence by flow cytometry in human thyrocytes incubated for 48 h with or without 1 mU/ml TSH, and transiently transfected or not with short interfering RNAs to NOX4 as described in Materials and methods.

Article Snippet: Antibody directed against the human NOX4 primary antibody was purchased from Novus Biologicals (Littleton, CO 80160, USA), and its specificity was tested with crude extracts prepared from HEK293 cells transiently transfected with the vector pcDNA3.1 containing or not human NOX4.

Techniques: Expressing, Incubation, Quantitative RT-PCR, Western Blot, Cytometry, Transfection

Journal: Endocrine-Related Cancer

Article Title: Intracellular expression of reactive oxygen species-generating NADPH oxidase NOX4 in normal and cancer thyroid tissues

doi: 10.1677/erc-09-0175

Figure Lengend Snippet: Figure 3 Comparative expression of NOX4 and p22 phox genes in human thyroid tissues analyzed by real-time quantitative reverse transcription PCR. Data are expressed as mRNA relative expression levels determined as X fold of calibrator corresponding to a pool of thyroid tissue samples and cell lines. Expression values (y-axis) are displayed on based 2 logarithmic scale. (A) Comparative expression of NOX4 and p22phox genes in several human thyroid tissue types: nontumoral (NT); follicular thyroid adenomas (FTA); follicular thyroid carcinomas (FTC); papillary thyroid carcinoma (PTC); and anaplastic thyroid carcinoma (ATC); (Table): distribution of mRNA relative expression levels of NOX4 and p22phox according to the histological type. Nb, number of cases in each histological group; Min/Max, minimum and maximum of mRNA relative expression levels in each histological group l; O75%Perc.NT, number of cases (percentage) displaying a mRNA relative expression level higher than the 75% percentile expression level of normal tissues; OMax of NT, number of cases (percentage) displaying a mRNA relative expression level higher than the higher expression level in normal tissues. (B) Comparative expression of NOX4 and p22phox genes in 15 paired samples of normal and tumor tissues. (NT, paired normal counterpart; T, tumors, corresponding to 2 (FTA), 3 FTC, 9 PTC, and 1 ATC. Each value represents the mRNA relative expression level.

Article Snippet: Antibody directed against the human NOX4 primary antibody was purchased from Novus Biologicals (Littleton, CO 80160, USA), and its specificity was tested with crude extracts prepared from HEK293 cells transiently transfected with the vector pcDNA3.1 containing or not human NOX4.

Techniques: Expressing, Reverse Transcription

Journal: Endocrine-Related Cancer

Article Title: Intracellular expression of reactive oxygen species-generating NADPH oxidase NOX4 in normal and cancer thyroid tissues

doi: 10.1677/erc-09-0175

Figure Lengend Snippet: Figure 4 Correlation between NOX4 and p22phox mRNA levels in normal and tumor tissues. Correlation between expression profiles was studied using Spearman’s rank-order correlation coefficient. The level of significance chosen was 5%.

Article Snippet: Antibody directed against the human NOX4 primary antibody was purchased from Novus Biologicals (Littleton, CO 80160, USA), and its specificity was tested with crude extracts prepared from HEK293 cells transiently transfected with the vector pcDNA3.1 containing or not human NOX4.

Techniques: Expressing

Journal: Endocrine-Related Cancer

Article Title: Intracellular expression of reactive oxygen species-generating NADPH oxidase NOX4 in normal and cancer thyroid tissues

doi: 10.1677/erc-09-0175

Figure Lengend Snippet: Figure 5 NOX4 protein expression in human thyroid tissues. (A) normal thyroid tissue. Note intracytoplasmic granular staining of NOX4 (arrows) on a diffusely stained background; FL, follicular lumen; magnification, !1000. (B) DUOX immunostaining is essentially localized at the apical membrane (arrow). Note faint, diffuse, intracytoplasmic staining. FL, follicular lumen. Magnification, !1000. (C) follicular hyperplasia. NOX4-positive intraluminal so-called ‘Sanderson pollster’ (solid arrow). Note negative flat quiescent cells in the same follicle (thin arrow). Magnification, !200. (D–F) NOX4, CD34 and NIS immunostainings on serial sections from the same follicle at the junction of activated columnar cells (solid arrows) and flat quiescent cells (thin arrows). Note on (E) that activated columnar cells are pseudostratified and present nuclei much larger than flat cells. Moreover, these cells are in contact to vessel endothelium, in contrast to flat cells. NOX4 and NIS display a strong immunostaining in large activated cells (solid arrows), and a negative or weak signal in quiescent flat cells (thin arrows). In vessels, red blood cells display pseudo-peroxidase activity, which has not been removed. Magnification, !400. (G) Follicular thyroid adenoma. Small follicles present a larger number of NOX4-positive cells than macrofollicles. Magnification, !200. (H) Papillary carcinoma. All tumor cells are NOX4 positive. Most staining is localized at the apical side, but some staining can be seen at the basal part (arrows). Magnification, !400. (I) Poorly differentiated follicular carcinoma. Diffuse staining is seen in all tumor cells. Magnification, !200.

Article Snippet: Antibody directed against the human NOX4 primary antibody was purchased from Novus Biologicals (Littleton, CO 80160, USA), and its specificity was tested with crude extracts prepared from HEK293 cells transiently transfected with the vector pcDNA3.1 containing or not human NOX4.

Techniques: Expressing, Staining, Immunostaining, Membrane, Activity Assay

Journal: Endocrine-Related Cancer

Article Title: Intracellular expression of reactive oxygen species-generating NADPH oxidase NOX4 in normal and cancer thyroid tissues

doi: 10.1677/erc-09-0175

Figure Lengend Snippet: Figure 6 p22phox protein expression in human thyroid tissues. (A) Thyroid nodular hyperplasia. As observed for NOX4, staining of p22phox is heterogeneous. Magnification, !200. (B) Follicular thyroid adenoma. p22phox immunostaining is essentially localized in activated columnar cells. Magnification, !100. (C–F) NOX4 and p22phox immunostaining on serial sections from the same tumoral region in poorly differentiated follicular carcinoma (PDFC) (magnification !100) and in papillary thyroid carcinoma (magnification !400) respectively. All tumor cells are positive and display the same staining profile. Note a strong p22phox immunostaining in macrophages corresponding to the expression of the NOX2–p22phox complex.

Article Snippet: Antibody directed against the human NOX4 primary antibody was purchased from Novus Biologicals (Littleton, CO 80160, USA), and its specificity was tested with crude extracts prepared from HEK293 cells transiently transfected with the vector pcDNA3.1 containing or not human NOX4.

Techniques: Expressing, Staining, Immunostaining

Journal: Life sciences

Article Title: Nox4 contributes to the hypoxia-mediated regulation of actin cytoskeleton in cerebrovascular smooth muscle.

doi: 10.1016/j.lfs.2016.08.018

Figure Lengend Snippet: Fig. 2. Hypoxia upregulated the expression of Nox4 and iNOS isoform in HBVSMC. HBVSMC were serum starved overnight, and then exposed to 30 min hypoxia/45 min reoxygenation. Nox4, Nox2, Nox1, iNOS and nNOS expressions were determined by Western blot. Hypoxia upregulated Nox4 (A) and iNOS (D) expression compared to normoxic conditions. Hypoxia had no effect on Nox2 (B) Nox1 (C) and nNOS (E) expression in HBVSMC (*p b 0.05, ***p b 0.001 vs normoxia, n = 4–6).

Article Snippet: Optimization experiments showed that A-033 program and 300 nM of Nox4 siRNA (OriGene Technologies, Rockville, MD) gave the maximum transfection efficacy for HBVSMC.

Techniques: Expressing, Western Blot

Journal: Life sciences

Article Title: Nox4 contributes to the hypoxia-mediated regulation of actin cytoskeleton in cerebrovascular smooth muscle.

doi: 10.1016/j.lfs.2016.08.018

Figure Lengend Snippet: Fig. 3. Silencing Nox4 expression in vitro. Nox4 expression was silenced in HBVSMC using electroporation-mediated siRNA delivery. (A) Representative Ct values and the statistical analysis for the various groups. Hypoxia increased Nox4 mRNA expression, and Nox4 silencing nullified this effect (n = 3). (B) Representative western blots of Nox4 and actin. Nox4 silencing reduced the expression of Nox4 compared to scrambled under normoxic condition, and blunted the increase in Nox4 expression after hypoxia (n = 3). (C, D) Representative western blots of Nox2 and Nox1. Nox4 silencing had no effect on the expression of both Nox2 and Nox1 compared to scrambled under normoxic conditions (n = 3). (*p b 0.01 disease treatment interaction).

Article Snippet: Optimization experiments showed that A-033 program and 300 nM of Nox4 siRNA (OriGene Technologies, Rockville, MD) gave the maximum transfection efficacy for HBVSMC.

Techniques: Expressing, In Vitro, Electroporation, Western Blot

Journal: Life sciences

Article Title: Nox4 contributes to the hypoxia-mediated regulation of actin cytoskeleton in cerebrovascular smooth muscle.

doi: 10.1016/j.lfs.2016.08.018

Figure Lengend Snippet: Fig. 4. Nox4 silencing restored superoxide and nitrotyrosine levels in HBVSMCs after hypoxia. Nox4 expression was silenced in HBVSMC using electroporation-mediated siRNA delivery. Cells were switched to overnight serum-free media, and then exposed to 30 min hypoxia/45 min reoxygenation. HBVSMC were fixed and stained for F actin with Oregon Green 488- conjugated phalloidin fluorescent probe and for nitrotyrosine with an anti-nitrotyrosine primary antibody and an IgG conjugated to Cy5 secondary antibody. Nitrotyrosine levels were detected by immunoblotting and immunostaining. (A) Silencing Nox4 expression blunted the increase in nitrotyrosine levels after hypoxia compared to scrambled RNA (n = 3–4). (B) Representative confocal microscopy images of HBVSMC stained for F actin cytoskeletal filaments (green) and colocalization of actin filaments with nitrotyrosine (red) are shown on top. Silencing Nox4 expression restored nitrotyrosine levels after hypoxia compared to scrambled RNA (n = 3). (C) Effect of hypoxia and Nox4 silencing on superoxide formation were detected using fluorometric assay of dihydroethidium (DHE). Superoxide formation was significantly increased after hypoxia and Nox4 silencing levels reduced it back to normal (n = 3–5). (D) Inhibiting iNOS with 1400 W dihydrochloride abolished the increase in nitrotyrosine levels after hypoxia compared to the vehicle (n = 5–9) (post hoc Bonferroni analyses *p b 0.05 vs scRNA normoxia, Nox4 siRNA normoxia and hypoxia, **p b 0.01 vs scRNA normoxia, ***p b 0.001 vs scRNA hypoxia, άp b 0.05 vs scRNA normoxia and #p b 0.01 vs scRNA hypoxia, **p b 0.01 disease treatment interaction).

Article Snippet: Optimization experiments showed that A-033 program and 300 nM of Nox4 siRNA (OriGene Technologies, Rockville, MD) gave the maximum transfection efficacy for HBVSMC.

Techniques: Expressing, Electroporation, Staining, Western Blot, Immunostaining, Confocal Microscopy

Journal: Life sciences

Article Title: Nox4 contributes to the hypoxia-mediated regulation of actin cytoskeleton in cerebrovascular smooth muscle.

doi: 10.1016/j.lfs.2016.08.018

Figure Lengend Snippet: Fig. 5. Silencing Nox4 expression restored F actin levels in HBVSMCs after hypoxia. Nox4 expression was silenced in HBVSMC using electroporation-mediated siRNA delivery. Cells were switched to overnight serum-free media, and then exposed to 30 min hypoxia/45 min reoxygenation. F:G actin were determined by western blot. In another group, HBVSMC were fixed and stained for F actin with Oregon Green 488-conjugated phalloidin fluorescent probe. (A) Representative western blot of F and G actin is shown on top. Silencing Nox4 expression significantly increased F:G actin after hypoxia compared to cells treated with scrambled RNA (n = 4). (B) Analysis of confocal microscopy images of F actin (green) showed that silencing Nox4 expression blunted the decrease of F actin after hypoxia(n = 3). (post hoc Bonferroni analyses *p b 0.05 vs scRNA hypoxia, άp b 0.05 vs scRNA normoxia).

Article Snippet: Optimization experiments showed that A-033 program and 300 nM of Nox4 siRNA (OriGene Technologies, Rockville, MD) gave the maximum transfection efficacy for HBVSMC.

Techniques: Expressing, Electroporation, Western Blot, Staining, Confocal Microscopy

Journal: The EMBO Journal

Article Title: Nox4 regulates InsP 3 receptor‐dependent Ca 2+ release into mitochondria to promote cell survival

doi: 10.15252/embj.2019103530

Figure Lengend Snippet: A, B Quantification of Nox4 mRNA levels (A) and protein levels (B) in cultured rat cardiomyocytes after increasing degrees of serum starvation as compared to serum‐replete cells (15% serum). n = 4 cell preparations/group. A representative immunoblot for Nox4 and tubulin as a loading control is shown in panel B. C Cell death after 48 h serum starvation in cardiomyocytes in which Nox4 was depleted by an adenoviral shRNA (Ad.shNox4) as compared to cells infected with a control vector (Ad.Ctl). n = 6/group. The representative immunoblot shows reduction in Nox4 protein levels after shRNA‐mediated knockdown. D Lactate dehydrogenase (LDH) activity in cell supernatants. Staurosporine (Stauro, 1 μmol/l) was used to induce cell death by apoptosis. n = 4 cell preparations/group. E Levels of high mobility group box 1 protein (HMGB1) in cell supernatants. n = 3/group. A representative immunoblot is shown at the top. F Protein levels of cytochrome c in the cytoplasmic fractions of cardiomyocytes. n = 4/group. A representative immunoblot is shown at the top. The cellular membrane fraction (Mem) containing mitochondria was used as a positive control. G Percentage of Nox4‐depleted and control cells with depolarized mitochondria, quantified by flow cytometry. n = 3 cell preparations/group (100,000 cells per experiment). H Effect of cyclosporin A (CsA, 1 μmol/l) on the percentage of cells with depolarized mitochondria. n = 6–8/group. I Effect of cyclosporin A (CsA, 1 μmol/l) on cell death in serum‐starved cardiomyocytes. n = 4/group. J Cell death in serum‐starved Nox4 knockout MEFs (Nox4KO) and wild‐type MEFs (WT). Cat = PEG‐catalase (500 U/ml). Nox4KO MEFs were transfected either with active Nox4 or a catalytic inactive Nox4 mutant, Nox4 P437H (Mut). n = 6–12/group. K HMGB1 levels in the supernatants of serum‐starved MEFs in similar experiments to those in (J). n = 4/group. A representative immunoblot is shown to the top. M Percentage of MEFs with depolarized mitochondria. n = 6/group. Data information: Data are mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001 among compared groups or vs. control. # P < 0.05; #### P < 0.0001 vs. Nox4KO. (C and G), 2‐way ANOVA; all other panels, 1‐way ANOVA. Source data are available online for this figure.

Article Snippet: Lentiviruses expressing a short hairpin sequence targeted against human Nox4 or a scramble short hairpin sequence were purchased from OriGene (Rockville, MD, USA; Cat. no #TL302911V) and used to infect hiPSC‐CM at an MOI of 10.

Techniques: Cell Culture, Western Blot, Control, shRNA, Infection, Plasmid Preparation, Knockdown, Activity Assay, Membrane, Positive Control, Flow Cytometry, Knock-Out, Transfection, Mutagenesis

Journal: The EMBO Journal

Article Title: Nox4 regulates InsP 3 receptor‐dependent Ca 2+ release into mitochondria to promote cell survival

doi: 10.15252/embj.2019103530

Figure Lengend Snippet: A Basal mitochondrial calcium levels assessed in serum‐starved rat cardiomyocytes using a mitochondrial‐targeted cameleon CFP/YFP FRET probe. Nox4 was depleted with silencing RNAs (siRNAs) or cells were transfected with a control scrambled siRNA (siScr). Representative photomicrographs are shown at the top. The spectrum color scale represents the ratio of emitted fluorescence (YFP/CFP). The mean changes in Nox4‐depleted cells as compared to scrambled control (dotted line) are shown at the bottom. The representative immunoblot shows depletion of Nox4 protein levels. Scale bars: 10 μm. n = 3/group (with > 50 cells per individual experiment). B Basal mitochondrial calcium levels in WT and Nox4KO MEFs after serum starvation. Representative photomicrographs are shown at the top. The spectrum color scale represents the ratio of emitted fluorescence (YFP/CFP). Mean data at the bottom. Scale bars: 10 μm. n = 3/group (with > 100 cells per individual experiment). C, D Changes in mitochondrial calcium levels in serum‐starved WT and Nox4KO MEFs after the addition of histamine (100 μmol/l, C) or ATP (100 μmol/l, D). n = 3/group (with > 30 cells per individual experiment). E, F Changes in ER calcium levels measured with an ER‐targeted cameleon probe in serum‐starved WT and Nox4KO MEFs after the addition of histamine (100 μmol/l, E) or ATP (100 μmol/l, F). n = 3/group (with > 30 cells per individual experiment). G Peak increase in mitochondrial calcium levels response in response to histamine (100 μmol/l) in WT MEFs with or without treatment with PEG‐catalase (Cat), and in Nox4KO MEFs with or without transfection with active Nox4 or a catalytically inactive Nox4 P437H mutant (Mut). n = 3/group (with > 30 cells per individual experiment). H Representative time course of histamine‐induced changes in mitochondrial calcium for the experiments shown in (G). Data information: Data are mean ± SEM. ** P < 0.01; **** P < 0.0001 among compared groups or vs. control. #### P < 0.0001 vs. Nox4KO. (A, B): Student's t ‐test; (C–H): 2‐way repeated measures ANOVA; (I): 1‐way ANOVA.

Article Snippet: Lentiviruses expressing a short hairpin sequence targeted against human Nox4 or a scramble short hairpin sequence were purchased from OriGene (Rockville, MD, USA; Cat. no #TL302911V) and used to infect hiPSC‐CM at an MOI of 10.

Techniques: Transfection, Control, Fluorescence, Western Blot, Mutagenesis

Journal: The EMBO Journal

Article Title: Nox4 regulates InsP 3 receptor‐dependent Ca 2+ release into mitochondria to promote cell survival

doi: 10.15252/embj.2019103530

Figure Lengend Snippet: A, B Time course of changes in mitochondrial calcium levels in serum‐starved WT and Nox4KO MEFs after treatment with histamine (Hist, A) or ATP (B). Images show the YFP/CFP ratio; agonists were added at t = 15 s. Related to Fig C and D, respectively. Scale bars: 10 μm. C, D Time course of changes in ER calcium levels in serum‐starved WT and Nox4KO MEFs after treatment with histamine (C) or ATP (D). Images show YFP/CFP ratio; agonists added at t = 15 s. Related to Fig E and F, respectively. Scale bars: 10 μm. E, F Time course of changes in cytosolic calcium levels in serum‐starved WT and Nox4KO MEFs after treatment with histamine (E) or ATP (F). Images show Fluo4 fluorescence. Related to Fig G and H, respectively. Scale bars: 10 μm. G Time course of histamine‐induced changes in mitochondrial calcium levels in serum‐starved WT and Nox4KO MEFs as in (A). Cat = PEG‐catalase. Nox4KO MEFs were transfected with active Nox4 or a catalytically inactive mutant (Mut). Relates to Fig I and J. Scale bars: 10 μm.

Article Snippet: Lentiviruses expressing a short hairpin sequence targeted against human Nox4 or a scramble short hairpin sequence were purchased from OriGene (Rockville, MD, USA; Cat. no #TL302911V) and used to infect hiPSC‐CM at an MOI of 10.

Techniques: Fluorescence, Transfection, Mutagenesis

Journal: The EMBO Journal

Article Title: Nox4 regulates InsP 3 receptor‐dependent Ca 2+ release into mitochondria to promote cell survival

doi: 10.15252/embj.2019103530

Figure Lengend Snippet: A, B Time course of histamine (Hist)‐induced changes in mitochondrial calcium levels in serum‐starved WT and Nox4 KO MEFs. TMRE + cells shown in (A) and TMRE − cells in (B). Sequential images to the right show YFP/CFP ratio, with quantification presented in the graphs. n = 3/group (with > 30 cells per individual experiment). Scale bars: 10 μm. C Peak histamine‐induced increase in mitochondrial calcium levels in serum‐starved WT and Nox4 KO MEFs with or without treatment with bongkrekic acid (BA) to inhibit the mPTP. n = 3/group (with > 30 cells per individual experiment). D Effect of caffeine or histamine on subcellular calcium levels in serum‐starved cardiomyocytes depleted of Nox4 or treated with a control scrambled siRNA (siScr). Percent changes in peak calcium levels are shown. n = 3/group (with > 30 cells per individual experiment). Data information: Data are mean ± SEM. ** P < 0.01; *** P < 0.001; **** P < 0.0001 among compared groups or vs. control. (A,B): 2‐way repeated measures ANOVA; (C): 1‐way ANOVA; (D): unpaired t ‐test.

Article Snippet: Lentiviruses expressing a short hairpin sequence targeted against human Nox4 or a scramble short hairpin sequence were purchased from OriGene (Rockville, MD, USA; Cat. no #TL302911V) and used to infect hiPSC‐CM at an MOI of 10.

Techniques: Control

Journal: The EMBO Journal

Article Title: Nox4 regulates InsP 3 receptor‐dependent Ca 2+ release into mitochondria to promote cell survival

doi: 10.15252/embj.2019103530

Figure Lengend Snippet: Immunoblotting of subcellular fractions from serum‐starved rat cardiomyoblasts (H9c2 cells). Input: homogenate before fractionation; Cyto: cytosol; ER: endoplasmic reticulum; Crude M: crude mitochondrial fraction, also containing MAM; Pure Mito: mitochondria; MAM: mitochondria‐associated ER membranes. Cytochrome c (Cyt c) was used as a mitochondrial marker, VDAC as an outer mitochondrial membrane marker, calnexin as an ER marker, Sigma1R and FACL4 as MAM markers, and tubulin as a cytosolic marker. Immunoblotting of subcellular fractions from rat heart. Immunoblotting of subcellular fractions from rat kidney. Electron micrographs showing subcellular localization of Nox4 in wild‐type (WT) and Nox4KO mouse hearts, and human‐induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CM). hiPSC‐CM were depleted of Nox4 using a lentiviral shRNA (Nox4KD) or infected with a control lentivirus. FACL4 was used as a MAM marker. Arrowheads show peri‐mitochondrial localization of Nox4 and FACL4, in the proximity of ER cisternae and ribosomes. M = mitochondria, ER = endoplasmic reticulum, T = T‐tubule. Scale bars: 100 nm. Source data are available online for this figure.

Article Snippet: Lentiviruses expressing a short hairpin sequence targeted against human Nox4 or a scramble short hairpin sequence were purchased from OriGene (Rockville, MD, USA; Cat. no #TL302911V) and used to infect hiPSC‐CM at an MOI of 10.

Techniques: Western Blot, Fractionation, Marker, Membrane, Derivative Assay, shRNA, Infection, Control

Journal: The EMBO Journal

Article Title: Nox4 regulates InsP 3 receptor‐dependent Ca 2+ release into mitochondria to promote cell survival

doi: 10.15252/embj.2019103530

Figure Lengend Snippet: Effect of serum starvation on Nox4 protein levels in WT MEFs. FACL4 and VDAC used as MAM markers. n = 3/group. Simplified photomicrographs of proximity ligation studies in WT MEFs under serum‐replete and serum‐starved conditions (as in Fig ). Nuclei are in blue, while yellow dots denote co‐localization of proteins. Quantification of the number of dots/cell is shown to the right. Proximity was tested for FACL4/Nox4. Scale bars: 10 μm. n = 3/group (with > 50 cells/individual experiment). Data information: Data are mean ± SEM. ** P < 0.01; *** P < 0.001 compared to control. Unpaired t ‐test. Source data are available online for this figure.

Article Snippet: Lentiviruses expressing a short hairpin sequence targeted against human Nox4 or a scramble short hairpin sequence were purchased from OriGene (Rockville, MD, USA; Cat. no #TL302911V) and used to infect hiPSC‐CM at an MOI of 10.

Techniques: Ligation, Control

Journal: The EMBO Journal

Article Title: Nox4 regulates InsP 3 receptor‐dependent Ca 2+ release into mitochondria to promote cell survival

doi: 10.15252/embj.2019103530

Figure Lengend Snippet: Simplified photomicrographs of proximity ligation studies in WT and Nox4KO MEFs, showing cell borders, nuclei (blue), and yellow dots corresponding to co‐localization of proteins. Quantification of the number of dots/cell in each condition is shown to the right. Proximity was tested for the following protein couples: FACL4/Nox4, InsP 3 R/Nox4, and FACL4/InsP 3 R. Scale bars: 10 μm. n = 3/group (with > 30 cells/individual experiment). Controls are shown in Appendix Fig S4B and the original unsimplified photographs in Appendix Fig S4E . Simplified photomicrographs of proximity ligation staining in rat cardiomyocytes transfected with Nox4 siRNA or a scrambled control (siScr), with quantification as in (A). Proximity was tested for the FACL4/Nox4 protein couple. Scale bars: 10 μm. n = 3/group (with > 30 cells/individual experiment). Controls and other protein couples are shown in Appendix Fig S4C and the original unsimplified photographs in Appendix Fig S4F . Simplified photomicrographs of proximity ligation staining in hiPSC‐CM (human cardiomyocytes) transduced with Nox4 shRNA or scrambled control, with quantification as in (A). Proximity was tested for the FACL4/Nox4 protein couple. Scale bars: 10 μm. n = 3/group (with > 30 cells/individual experiment). Controls and other protein couples are shown in Appendix Fig S4D and the original unsimplified photographs in Appendix Fig S4G . Data information: Data are mean ± SEM. **** P < 0.0001 vs. corresponding control (unpaired t ‐test).

Article Snippet: Lentiviruses expressing a short hairpin sequence targeted against human Nox4 or a scramble short hairpin sequence were purchased from OriGene (Rockville, MD, USA; Cat. no #TL302911V) and used to infect hiPSC‐CM at an MOI of 10.

Techniques: Ligation, Staining, Transfection, Control, Transduction, shRNA

Journal: The EMBO Journal

Article Title: Nox4 regulates InsP 3 receptor‐dependent Ca 2+ release into mitochondria to promote cell survival

doi: 10.15252/embj.2019103530

Figure Lengend Snippet: Immunoblotting of subcellular fractions from serum‐starved cardiomyoblasts for InsP R, Akt and PP2a, as in Fig A. Fractionation of crude mitochondrial fractions (containing mitochondria and MAM) from WT MEFs on a sucrose gradient (5–60%) and immunoblotting for relevant proteins. Fraction size: 200 μl. Representative of 3 independent experiments. Immunoblotting for phosphorylated and total Akt or InsP R protein in crude mitochondrial fractions from serum‐starved WT and Nox4KO MEFs. FACL4 was used as a loading control for the MAM. InsP R was first immunoprecipitated, and then, the precipitate was immunoblotted for total InsP R and for the phosphorylated Akt substrate motif RXRXX(pS/T) (p‐InsP R). Representative immunoblots are shown at the top and mean data at the bottom. Cat = PEG‐catalase. Nox4KO MEFs were transfected either with active Nox4 or a Nox4 P437H mutant (Mut). n = 3/group. ROS levels indexed in MEFs using H 2 O 2 specific HyPer probes targeted to the MAM (FACL4 HyPer) or the cytosol (Cyto HyPer). Imaging was performed after serum starvation for 12 h. Representative photomicrographs of the fluorescence ratio are shown to the right and mean data for changes in fluorescence ratio presented to the left. The signal obtained using corresponding ROS‐insensitive SypHer probes was used to correct for any pH‐induced changes in fluorescence; the change in fluorescence ratio between HyPer and corresponding SypHer probe (ΔR) for each condition is reported. n = 5 independent cell preparations/group, with at least 20 cells imaged/preparation. Scale bars: 10 μm. Data information: Data are mean ± SEM. *** P < 0.001; **** P < 0.0001 among compared groups or vs. control. ### P < 0.001; ## P < 0.01 compared to Nox4KO (1‐way ANOVA). Source data are available online for this figure.

Article Snippet: Lentiviruses expressing a short hairpin sequence targeted against human Nox4 or a scramble short hairpin sequence were purchased from OriGene (Rockville, MD, USA; Cat. no #TL302911V) and used to infect hiPSC‐CM at an MOI of 10.

Techniques: Western Blot, Fractionation, Control, Immunoprecipitation, Transfection, Mutagenesis, Imaging, Fluorescence

Journal: The EMBO Journal

Article Title: Nox4 regulates InsP 3 receptor‐dependent Ca 2+ release into mitochondria to promote cell survival

doi: 10.15252/embj.2019103530

Figure Lengend Snippet: Mitochondrial ROS levels in serum‐starved WT and Nox4 KO MEFs indexed using a mitochondrial‐targeted HyPer probe (Mito HyPer). Representative photomicrographs of the fluorescence ratio are shown to the left and mean data for changes in fluorescence ratio to the right. The corresponding ROS‐insensitive SypHer probe was used to correct for any pH‐induced changes in fluorescence. n = 3 independent cell preparations/group, with at least 50 cells imaged/preparation. Scale bars: 10 μm. Effect of Mito‐TEMPO on the phosphorylation of InsP 3 R and Akt in serum‐starved WT MEFs. InsP 3 R was first immunoprecipitated and then the precipitate was immunoblotted for total InsP 3 R and for the phosphorylated Akt substrate motif RXRXX(pS/T) (p‐InsP 3 R). FACL4 was used as a loading control for the MAM. Mean data shown to the bottom. n = 4/group. Effect of Mito‐TEMPO on histamine‐induced changes in mitochondrial calcium in serum‐starved cells. Representative images of YFP/CFP ratio are shown to the right. n = 3/group (with > 30 cells per individual experiment). Scale bars: 10 μm. Ser/Thr phosphatase activity measured in crude mitochondrial fractions of MEFs in the presence or absence of okadaic acid (OA, 10 nmol/l; specific for PP2a inhibition) or calyculin A (Caly, 60 nmol/l; which inhibits PP1 and PP2a) or H 2 O 2 (500 μmol/l). PP2a activity was calculated as the difference between control and OA, and PP1+PP2a activity as the difference between control and Caly. n = 5/group. **** P < 0.0001 compared to respective control; # P < 0.05, ### P < 0.001, #### P < 0.0001 comparing Nox4KO to respective WT group. Effect of treatment of cells with PEG‐catalase (Cat) or transfection of Nox4KO cells with active Nox4 or mutant Nox4 P437H (Mut) on Ser/Thr phosphatase activity. n = 4/group. *** P < 0.001, **** P < 0.0001 compared to WT control group; #### P < 0.0001 compared to Nox4KO group. Data information: Data are mean ± SEM. (A,B): unpaired t ‐test; (C): 2‐way repeated measures ANOVA; (D, E): 1‐way ANOVA.

Article Snippet: Lentiviruses expressing a short hairpin sequence targeted against human Nox4 or a scramble short hairpin sequence were purchased from OriGene (Rockville, MD, USA; Cat. no #TL302911V) and used to infect hiPSC‐CM at an MOI of 10.

Techniques: Fluorescence, Phospho-proteomics, Immunoprecipitation, Control, Activity Assay, Inhibition, Transfection, Mutagenesis

Journal: The EMBO Journal

Article Title: Nox4 regulates InsP 3 receptor‐dependent Ca 2+ release into mitochondria to promote cell survival

doi: 10.15252/embj.2019103530

Figure Lengend Snippet: Schematic of putative mechanism underlying Nox4‐mediated regulation of calcium transfer via InsP 3 R to mitochondria. Akti: Akt inhibitor; XeC: xestospongin C (InsP 3 R blocker). Basal mitochondrial calcium levels in serum‐starved WT and Nox4KO MEFs in the presence or absence of Akti (1 μmol/l) normalized versus WT MEF control. n = 6/group with > 20 cells imaged per experiment. Effect of Akti (1 μmol/l) or XeC (1 μmol/l) on histamine‐induced changes in mitochondrial calcium levels in MEFs. The time course of changes in calcium levels is shown on the left and the mean data for peak mitochondrial calcium levels on the right. n = 3/group with > 40 cells imaged per experiment. Quantification of cell death in serum‐starved WT and Nox4KO MEFs in the absence or presence of Akti or XeC. n = 6/group. Cardiac troponin I (cTnI) levels as a marker of cardiomyocyte necrosis in the perfusate of isolated WT and Nox4KO hearts subjected to ischemia–reperfusion (I/R). XeC was added at a final concentration of 2 μmol/l prior to ischemia. n = 6–7/group. Immunoblotting for the phosphorylation levels of Akt and InsP 3 R in crude mitochondrial fractions from WT and Nox4KO hearts after I/R. InsP 3 R was first immunoprecipitated, and then, the precipitate was immunoblotted for total InsP 3 R and for the phosphorylated Akt substrate motif RXRXX(pS/T) (p‐InsP 3 R). Mean data shown to the right. n = 3/group. Cardiac left ventricular contractile function in isolated Langendorff‐perfused WT and Nox4KO hearts at baseline (Basal) and then after I/R. Hearts were treated with XeC (1 μmol/l) or vehicle control for 20 min prior to ischemia. RPP, heart rate × left ventricular pressure product; DEVP, left ventricular developed pressure. n = 6–7/group. Data information: Data are mean ± SEM. * P < 0.05, ** P < 0.01; **** P < 0.0001 among compared groups or vs. control. # P < 0.05; #### P < 0.0001 among different groups within the same treatment or among different treatments within the same genotype. (B,G): 1‐way ANOVA; (C‐E): 2‐way ANOVA; (F): unpaired t ‐test.

Article Snippet: Lentiviruses expressing a short hairpin sequence targeted against human Nox4 or a scramble short hairpin sequence were purchased from OriGene (Rockville, MD, USA; Cat. no #TL302911V) and used to infect hiPSC‐CM at an MOI of 10.

Techniques: Control, Marker, Isolation, Concentration Assay, Western Blot, Phospho-proteomics, Immunoprecipitation

Journal: The EMBO Journal

Article Title: Nox4 regulates InsP 3 receptor‐dependent Ca 2+ release into mitochondria to promote cell survival

doi: 10.15252/embj.2019103530

Figure Lengend Snippet: Changes in histamine‐induced mitochondrial calcium levels in serum‐starved WT and Nox4KO MEFs in the presence or absence of Akti or xestospongin C (XeC). Images show the YFP/CFP ratio at different time points. Relates to Fig C. Scale bars: 10 μm. Percentage of serum‐starved cardiomyocytes with depolarized mitochondria in the absence or presence of XeC. n = 9/group. Cell death in serum‐starved cardiomyocytes depleted of Nox4 or control cells, in the absence or presence of XeC. n = 7/group. Changes in histamine‐induced mitochondrial calcium levels in serum‐starved WT and Nox4KO MEFs in the presence or absence of okadaic acid, as in (A). Scale bars: 10 μm. n = 3/group (with > 50 cells per individual experiment). Cell death in serum‐starved WT and Nox4KO MEFs in the presence or absence of okadaic acid. n = 5/group. Indices of cardiac contractile function in perfused WT and Nox4KO hearts at baseline (Basal) and then after 25 min ischemia/30 min reperfusion (recovery). Hearts were treated with XeC (1 μmol/l) or vehicle control for 20 min prior to ischemia. HR = heart rate; d P /d t max and d P /d t min = maximal rate of rise and fall, respectively, of left ventricular (LV) pressure; EDP = LV end‐diastolic pressure. n = 6–7/group. Data information: Data are mean ± SEM. * P < 0.05; *** P < 0.001; **** P < 0.0001 among compared groups or vs. control. # P < 0.05, #### P < 0.0001 vs. Nox4KD or vehicle control. (B,C,E,F): 1‐way ANOVA; (D): paired t ‐test.

Article Snippet: Lentiviruses expressing a short hairpin sequence targeted against human Nox4 or a scramble short hairpin sequence were purchased from OriGene (Rockville, MD, USA; Cat. no #TL302911V) and used to infect hiPSC‐CM at an MOI of 10.

Techniques: Control

Journal: The EMBO Journal

Article Title: Nox4 regulates InsP 3 receptor‐dependent Ca 2+ release into mitochondria to promote cell survival

doi: 10.15252/embj.2019103530

Figure Lengend Snippet: During cellular stress, Nox4 at the MAM augments the level of Akt activation secondary to redox inhibition of PP2a. Activated Akt in turn phosphorylates InsP 3 R and leads to an inhibition of calcium flux from ER to mitochondria. Mitochondrial calcium is maintained at a low level, the mitochondrial membrane potential (ΔΨ m ) is preserved, and cells remain viable. When Nox4 is deficient (bottom panel), high PP2a activity dephosphorylates Akt and leads to a reduced level of InsP 3 R phosphorylation. This removes the restraint on calcium transfer from ER to mitochondria resulting in increased mitochondrial calcium levels, triggering of the mPT, loss of ΔΨ m, and eventual cell death.

Article Snippet: Lentiviruses expressing a short hairpin sequence targeted against human Nox4 or a scramble short hairpin sequence were purchased from OriGene (Rockville, MD, USA; Cat. no #TL302911V) and used to infect hiPSC‐CM at an MOI of 10.

Techniques: Activation Assay, Inhibition, Membrane, Activity Assay, Phospho-proteomics