Journal: The Journal of Clinical Investigation

Article Title: Inositol-triphosphate 3-kinase B confers cisplatin resistance by regulating NOX4-dependent redox balance

doi: 10.1172/JCI124550

Figure Lengend Snippet: (A) Effect of ITPKB knockdown on the activity of ROS-regulating enzymes in KB-3-1cisR and A549cisR cells. Cells with or without ITPKB knockdown were treated with cisplatin (KB-3-1cisR, 5 μg/ml; A549cisR, 2 μg/ml), and activities of redox regulatory enzymes were measured after 12 hours. (B) Effect of NOX1/4 inhibitor GKT137831 on ROS level, NOX activity, and cell viability in ITPKB-knockdown cells in the presence of cisplatin. Cells were treated with GKT137831 (10 μM) and cisplatin as in A. (C) Effect of NOX4 knockdown on ROS level, NOX activity, and cell viability in ITPKB-knockdown cells in the presence of cisplatin. (D and E) Bcl-xL expression (D) and Bax localization (E) change upon GKT137831 treatment in ITPKB-knockdown cells. c, cytosol; m, mitochondria. (F–H) The rescue effect of NOX inhibitor on the tumor growth of ITPKB-knockdown KB-3-1cisR xenograft mice treated with cisplatin. Mice were treated with vehicle control, cisplatin (5 mg/kg), and GKT137831 (30 mg/kg) by i.p. injection twice a week from 3 days after xenograft. Tumor size (F), tumor weight (G), H2O2 level (H, top), NOX activity (H, middle), and ITPKB expression in tumors (H, bottom) are shown. Scale bars: 10 mm for F. Error bars represent SEM for F and SD for G (n = 7). Data are mean ± SD from 3 technical replicates of each sample and are representative of 2 independent biological experiments for A–E and H. Statistical analysis was performed by 2-way ANOVA for F and 1-way ANOVA for all other data (*P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.0001).

Article Snippet: Anti-NOX4 antibodies were obtained from ProSci (7927) for immunoblotting cell lysates and Abcam (ab133303/UOTR1B493) for immunofluorescence staining and immunoblotting of recombinant NOX4-DH.

Techniques: Activity Assay, Expressing, Injection

Journal: The Journal of Clinical Investigation

Article Title: Inositol-triphosphate 3-kinase B confers cisplatin resistance by regulating NOX4-dependent redox balance

doi: 10.1172/JCI124550

Figure Lengend Snippet: (A) Purification of the recombinant NOX4 dehydrogenase active domain (NOX4-DH). Top: Schematic representation of NOX4 domains. TM, transmembrane domain; FAD, flavin adenine dinucleotide binding domain; NADPH, NADPH binding domain. Bottom: Coomassie staining of purified recombinant NOX4-DH. (B) Thermal shift analyses of NOX4-DH incubated with increasing concentrations of IP4. Tm, melting temperature. (C) Thermal shift analyses of NOX4-DH incubated with 80 μM of the inositol metabolites PIP3, IP3, IP4, and IP5. Dissociation constant (KD) values for the interaction are shown. ND, not determined. (D) Interaction between IP4 and NOX4-DH was determined by Biacore surface plasmon resonance analysis and is shown as a KD value. (E and F) The activity of purified NOX4-DH was measured in the presence of increasing concentrations of IP4 (E) or 80 μM of different inositol metabolites (F). (G) Quantification of NADPH bound to NOX4 in the presence of increasing concentrations of IP4. FLAG-tagged NOX4-DH (30 μM) was incubated with NADPH (100 μM) and IP4 (0–100 μM). Amount of NADPH retained on immobilized NOX4-DH was determined by measurement of absorbance at A340 nm. Data are mean ± SD from 3 technical replicates of each sample for E–G. Data shown are representative of 2 (B–D) and 3 (E–G) independent biological experiments. Statistical analysis was performed by 1-way ANOVA (****P < 0.0001).

Article Snippet: Anti-NOX4 antibodies were obtained from ProSci (7927) for immunoblotting cell lysates and Abcam (ab133303/UOTR1B493) for immunofluorescence staining and immunoblotting of recombinant NOX4-DH.

Techniques: Purification, Recombinant, Binding Assay, Staining, Incubation, SPR Assay, Activity Assay

Journal: The Journal of Clinical Investigation

Article Title: Inositol-triphosphate 3-kinase B confers cisplatin resistance by regulating NOX4-dependent redox balance

doi: 10.1172/JCI124550

Figure Lengend Snippet: (A) BAMB-4 inhibits ITPKB activity in vitro (left) and in KB-3-1 cells (right). (B) Effect of BAMB-4 on ROS level, NOX activity, and cisplatin IC50 in cisplatin-treated cells with or without ITPKB knockout. Cells were treated with cisplatin (KB-3-1cisR and A2780cisR, 5 μg/ml; A549cisR, 2 μg/ml) and BAMB-4 (10 μM) for 12 hours, and ROS and NOX activity were examined. (C and D) Effect of BAMB-4 and/or cisplatin treatment on tumor growth (C) and tumor size (D) of ovarian cancer PDX mice. Mice were treated with cisplatin (5 mg/kg) and BAMB-4 (10 mg/kg) by i.p. injection twice a week after 42 days from xenograft. (E) Effect of BAMB-4 and/or cisplatin treatment on ITPKB activity, H2O2 level, and NOX activity in ovarian cancer PDX tumors is shown. Scale bars: 10 mm for C. Error bars represent SEM for C and SD for D (n = 6). Data are mean ± SD from 3 technical replicates of each sample and are representative of 2 independent biological experiments for A, B, and E. Statistical analysis was performed by 2-way ANOVA for C and 1-way ANOVA for all other data (*P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.0001). (F) Proposed model of ITPKB-mediated cisplatin resistance in human cancer. The ITPKB product IP4 inhibits NOX4 by competing with its cofactor NADPH for binding. IP4-induced NOX4 inhibition controls cisplatin-induced ROS, which provides a metabolic advantage to cancer cells that leads to cisplatin-resistant cancer cell survival and tumor growth.

Article Snippet: Anti-NOX4 antibodies were obtained from ProSci (7927) for immunoblotting cell lysates and Abcam (ab133303/UOTR1B493) for immunofluorescence staining and immunoblotting of recombinant NOX4-DH.

Techniques: Activity Assay, In Vitro, Knock-Out, Injection, Binding Assay, Inhibition

Journal: CNS Neuroscience & Therapeutics

Article Title: miR‐322 treatment rescues cell apoptosis and neural tube defect formation through silencing NADPH oxidase 4

doi: 10.1111/cns.13383

Figure Lengend Snippet: Expression of miR‐322 and NOX4 in NTDs caused by ATRA. (A) Morphology of normal and NTDs embryos at E9.5. (B) HE staining images of the neural tube transverse sections of normal and NTD embryos at E9.5. (C) Relative expression of miR‐322 was measured by qRT‐PCR using U6 as an internal control. (D) Western blot was performed to examine NOX4 levels using β‐actin as an internal control. (E) Representative IHC images of NOX4 expression in neural tube at E9.5 and semi‐quantitative analysis of the IHC results. (F) The relative expression of NOX4 mRNA was tested by qRT‐PCR using β‐actin as an internal control. (G) Representative immunofluorescence results of NOX4 (green) and the TUNEL signal (red) in E9.5 samples. DAPI (blue) was counterstained to reveal the nucleus. Statistical analysis of the fluorescence intensity and the percentage of TUNEL positive signal colocalized with strong NOX4 expression. The result represents three repeated experiments. “*,” “**,” and “***” represented P < .05, P < .01, and P < .001, respectively

Article Snippet: A nonfat milk solution (5%) was used for blocking before incubation with primary antibodies against NOX4 (1:1000, ROCKLAND), Bax (1:1000, Cell Signaling Technology), cleaved caspase‐3 (1:500, Cell Signaling Technology), or Bcl‐2 (1:1000, Sigma‐Aldrich) overnight at 4°C.

Techniques: Expressing, Staining, Quantitative RT-PCR, Control, Western Blot, Immunofluorescence, TUNEL Assay, Fluorescence

Journal: CNS Neuroscience & Therapeutics

Article Title: miR‐322 treatment rescues cell apoptosis and neural tube defect formation through silencing NADPH oxidase 4

doi: 10.1111/cns.13383

Figure Lengend Snippet: miR‐322 interacts with its target gene, NOX4. (A) Schematic representation of the NOX4 mRNA depicting miR‐322 binding sites in its 3’‐UTR. One predicted miR‐322 binding site (position 2437‐2460) is located in the 3’‐UTR of NOX4 mRNA. The effect of miR‐322 (B) and U6 (C) on the mRNA expression level at 48 hour after biotin‐miR‐322 transfection was detected using qRT‐PCR. (D) NOX4 mRNA level in the precipitates pulled down by biotin‐miR‐322 and biotin‐labeled negative control was detected using qRT‐PCR. (E) qRT‐PCR was used to detect the expression level of input NOX4 mRNA. All experiments were performed in triplicate. “**” and “****,” respectively, represented P < .01 and P < .0001, compared with the Bio‐con group (transfected with biotin‐labeled scrambled control miRNA)

Article Snippet: A nonfat milk solution (5%) was used for blocking before incubation with primary antibodies against NOX4 (1:1000, ROCKLAND), Bax (1:1000, Cell Signaling Technology), cleaved caspase‐3 (1:500, Cell Signaling Technology), or Bcl‐2 (1:1000, Sigma‐Aldrich) overnight at 4°C.

Techniques: Binding Assay, Expressing, Transfection, Quantitative RT-PCR, Labeling, Negative Control, Control

Journal: CNS Neuroscience & Therapeutics

Article Title: miR‐322 treatment rescues cell apoptosis and neural tube defect formation through silencing NADPH oxidase 4

doi: 10.1111/cns.13383

Figure Lengend Snippet: miR‐322 suppresses NOX4 translation. (A) Levels of reporter activity as measured by analysis of the 3’UTR‐Luc, BS‐Luc, and Mut‐Luc luciferase reporters after overexpression of miR‐322. Left, plasmid schematic of different chimeric firefly luciferase‐NOX4 reporters. Luc, luciferase; BS, miR‐322‐binding site; Mut, mutation. Right, the luciferase activities levels of 3’UTR‐Luc, BS‐Luc, and Mut‐Luc reporters. C17.2 cells were transfected with different NOX4‐luciferase reporter plasmids, together with miR‐322 mimic or negative control. Levels of firefly and Renilla luciferase activities were assayed 48 hour later. Results were normalized to the Renilla luciferase activity. (B) miR‐322 levels in cells transfected with the miR‐322 mimic. Protein abundance (C) and mRNA levels (D) of NOX4 after miR‐322 mimic transfection. (E) miR‐322 levels in cells transfected with the miR‐322 inhibitor. Effect of the miR‐322 inhibitor on NOX4 protein (F) and mRNA (G) expression. The assay was repeated for three times. “*” and “**” represented P < .05 and P < .01, respectively

Article Snippet: A nonfat milk solution (5%) was used for blocking before incubation with primary antibodies against NOX4 (1:1000, ROCKLAND), Bax (1:1000, Cell Signaling Technology), cleaved caspase‐3 (1:500, Cell Signaling Technology), or Bcl‐2 (1:1000, Sigma‐Aldrich) overnight at 4°C.

Techniques: Activity Assay, Luciferase, Over Expression, Plasmid Preparation, Binding Assay, Mutagenesis, Transfection, Negative Control, Quantitative Proteomics, Expressing

Journal: CNS Neuroscience & Therapeutics

Article Title: miR‐322 treatment rescues cell apoptosis and neural tube defect formation through silencing NADPH oxidase 4

doi: 10.1111/cns.13383

Figure Lengend Snippet: miR‐322 attenuates NOX4‐triggered cell apoptosis. (A) The protein expression of cleaved caspase‐3, Bax, Bcl‐2, and NOX4 was determined by Western blot using β‐actin as an internal control. (B) Cleaved caspase‐3, Bax, Bcl‐2, and NOX4 protein levels were determined by Western blot after cells were transfected with NOX4 and/or the miR‐322 mimic. (C) Representative images of the TUNEL assay. Cells were transfected with the NOX4 full‐length expression plasmid, and cotransected with miR‐322 mimic or control mimic. Apoptotic cells were labeled by the TUNEL reagents and all cell nuclei were stained by DAPI. These assays were repeated three times. * P < .05, ** P < .01, *** P < .001, **** P < .0001

Article Snippet: A nonfat milk solution (5%) was used for blocking before incubation with primary antibodies against NOX4 (1:1000, ROCKLAND), Bax (1:1000, Cell Signaling Technology), cleaved caspase‐3 (1:500, Cell Signaling Technology), or Bcl‐2 (1:1000, Sigma‐Aldrich) overnight at 4°C.

Techniques: Expressing, Western Blot, Control, Transfection, TUNEL Assay, Plasmid Preparation, Labeling, Staining

Journal: CNS Neuroscience & Therapeutics

Article Title: miR‐322 treatment rescues cell apoptosis and neural tube defect formation through silencing NADPH oxidase 4

doi: 10.1111/cns.13383

Figure Lengend Snippet: miR‐322 treatment alleviates cell apoptosis in ATRA‐induced NTD embryos. (A) Morphology of embryos from different groups after 48h of culture. (B) The number of NTDs and normally developed (normal) embryos. (C) qRT‐PCR was used to detect the expression level of miR‐322 using U6 as an internal control (n = 5). (D) Relative expression of NOX4, cleaved caspase‐3, Bax, and Bcl‐2 proteins was determined by Western blot using β‐actin as an internal control. (E) Representative images of the TUNEL assay. Apoptotic cells were labeled with TUNEL reagents, and all cell nuclei were stained by DAPI. agomiR‐322: miR‐322‐5p agomir. These assays were repeated three times. * P < .05, ** P < .01, *** P < .001, **** P < .0001

Article Snippet: A nonfat milk solution (5%) was used for blocking before incubation with primary antibodies against NOX4 (1:1000, ROCKLAND), Bax (1:1000, Cell Signaling Technology), cleaved caspase‐3 (1:500, Cell Signaling Technology), or Bcl‐2 (1:1000, Sigma‐Aldrich) overnight at 4°C.

Techniques: Quantitative RT-PCR, Expressing, Control, Western Blot, TUNEL Assay, Labeling, Staining

Journal: CNS Neuroscience & Therapeutics

Article Title: BMPER alleviates ischemic brain injury by protecting neurons and inhibiting neuroinflammation via Smad3‐Akt‐Nrf2 pathway

doi: 10.1111/cns.13782

Figure Lengend Snippet: BMPER inhibits microglial activation, adhesion molecule, and superoxide‐generating enzyme in MCAO model. (A) Immunohistochemistry staining of microglia activation marker Iba‐1 in penumbra area of MCAO mice receiving BMPER treatment. (B) Immunohistochemistry staining of adhesion molecule ICAM‐1 in penumbra area of MCAO mice receiving BMPER treatment. (C, D) Immunohistochemistry staining of superoxide‐generating enzymes NOX1 and NOX4 in penumbra area of MCAO mice receiving BMPER treatment. Data are expressed as mean ± SEM, n = 6. ** p < 0.01 by ANOVA followed by LSD‐t post hoc test. BMP, bone morphogenetic protein; BMPER, BMP‐binding endothelial regulator; MCAO, middle cerebral artery occlusion

Article Snippet: The primary antibodies used were as follows: TNF‐α (ab183218; Abcam), IL‐6 (ab214429; Abcam), IL‐1β (sc‐52012; Santa Cruz), Iba‐1 (sc‐32725; Santa Cruz), ICAM‐1 (ab119871; Abcam), NOX1 (bs‐3682R; Bioss), and NOX4 (bs‐1091R; Bioss).

Techniques: Activation Assay, Immunohistochemistry, Staining, Marker, Binding Assay

Journal: PLoS ONE

Article Title: Deficiency of NOX1 or NOX4 Prevents Liver Inflammation and Fibrosis in Mice through Inhibition of Hepatic Stellate Cell Activation

doi: 10.1371/journal.pone.0129743

Figure Lengend Snippet: Primer Sequences for Real-time.

Article Snippet: The NOX1 and NOX4 antibodies were purchased from Boster Biological Technology Company, Wuhan, China.

Techniques:

Journal: PLoS ONE

Article Title: Deficiency of NOX1 or NOX4 Prevents Liver Inflammation and Fibrosis in Mice through Inhibition of Hepatic Stellate Cell Activation

doi: 10.1371/journal.pone.0129743

Figure Lengend Snippet: (A) HSC proliferation is reduced in NOX1KO and NOX4KO mice after CCl 4 injury. HSC proliferation in liver was presented by PCNA immunofluorescence microscopy. Desmin is a specific marker of HSCs (Red). PCNA is a marker of proliferation (Green). Original magnification X10. (B) Proliferation was reduced in HSCs lacking NOX1 and NOX4 in response to PDGF (10 ng/ml) for 24 h. Proliferative HSCs were presented by Ki67 (Red) immunofluorescence microscopy. Nuclei were presented by DAPI (Blue). Original magnification X10. (C) Graph of PCNA-positive HSCs in vivo. (D) Graph of Ki67-positive HSCs in vitro.

Article Snippet: The NOX1 and NOX4 antibodies were purchased from Boster Biological Technology Company, Wuhan, China.

Techniques: Immunofluorescence, Microscopy, Marker, In Vivo, In Vitro

Journal: PLoS ONE

Article Title: Deficiency of NOX1 or NOX4 Prevents Liver Inflammation and Fibrosis in Mice through Inhibition of Hepatic Stellate Cell Activation

doi: 10.1371/journal.pone.0129743

Figure Lengend Snippet: Human livers from controls (N = 7) and patients with cirrhosis (N = 10) were analyzed by Sirius Red staining and by immunofluorescence with antibodies against NOX1 and NOX4. The graphs show the percent positive area of the staining or immunofluorescence expressed as mean+S.D., p<0.05 for each comparison of control vs. fibrosis.

Article Snippet: The NOX1 and NOX4 antibodies were purchased from Boster Biological Technology Company, Wuhan, China.

Techniques: Staining, Immunofluorescence, Comparison, Control

Journal: Antioxidants & Redox Signaling

Article Title: NADPH Oxidase NOX4 Is a Critical Mediator of BRAF V600E -Induced Downregulation of the Sodium/Iodide Symporter in Papillary Thyroid Carcinomas

doi: 10.1089/ars.2015.6616

Figure Lengend Snippet: Expression of NADPH oxidase NOX4 and its functional partner p22phox under the control of BRAFV600E oncogene. Time course expression of NOX4 and p22phox in BCPAP. (A) NOX4 and p22phox mRNA were quantified by real-time quantitative reverse transcription PCR (qRT-PCR). (B) NOX4 and p22phox proteins were analyzed by Western blot as described in the Materials and Methods section. Densitometric quantification of Nox4 and p22phox levels normalized to actin levels and presented as fold change compared with the basal level (24 h). (C) Intracellular ROS detection in BCPAP cells by DCF fluorescence using flow cytometry 48 h after knocking down of NOX4 or p22phox with specific siRNA (10 nM). The relative fluorescence units (RFU) are normalized to 100 in the control. (D) Immunoblot detection of NOX4 and p22phox in BCPAP cells after pharmacological inhibition of BRAFV600E oncogene by PLX4032 (10 μM). Densitometric quantification of Nox4 and p22phox levels normalized to actin levels and presented as fold change compared with vehicle-treated cells for 24 h. Student t-test is realized by comparing PLX versus DMSO for each corresponding time of the kinetic. Values are mean ± SE. *p < 0.05, **p < 0.01, and ***p < 0.001. ROS, reactive oxygen species.

Article Snippet: Immunodetection was performed as described previously ( 48 ) using as primary antibody anti-NOX4 (OriGene); anti-pSmad3 (ser 423) (Abcam); anti-Smad3 (Abcam); anti-p22 phox (Santa Cruz Biotechnology, Inc.), anti-NIS (Thermo Scientific); anti-BRAF V600E (Spring); anti-β-actin (Sigma-Aldrich), and anti-Vinculin (Abcam).

Techniques: Expressing, Functional Assay, Quantitative RT-PCR, Western Blot, Fluorescence, Flow Cytometry, Inhibition

Journal: Antioxidants & Redox Signaling

Article Title: NADPH Oxidase NOX4 Is a Critical Mediator of BRAF V600E -Induced Downregulation of the Sodium/Iodide Symporter in Papillary Thyroid Carcinomas

doi: 10.1089/ars.2015.6616

Figure Lengend Snippet: BRAFV600E oncogene upregulates NOX4 via TGF-β1 signaling pathway in papillary thyroid cancer cell line. (A) Quantitative detection of secreted and accumulated TGF-β1 in the extracellular medium of BCPAP cells using ELISA test. (B) Western blot analysis of total and phospho Smad3 proteins in BCPAP cells at indicated time of culture. Densitometric quantification of phospho Smad3 levels normalized to actin levels and presented as fold change compared with the basal level (24 h). (C) Immunoblot detection of Nox4, Smad3, and phospho-Smad3 proteins in BCPAP cells treated with SIS3 (10 μM). Densitometric quantification of Nox4 and phospho-Smad3 levels normalized to actin levels and presented as fold change compared with vehicle-treated cells or inhibitor-treated cells for 24 h. Student t-test is realized by comparing SIS3 versus DMSO for each corresponding time of the kinetic. (D) Inhibition of NOX4, p22phox, Smad3, and phospho-Smad3 protein expressions by the specific inhibitor of MAPK pathway U0126 (10 μM). Proteins were visualized by Western blot in BCPAP cells treated with U0126 for 48 h. Densitometric quantification of Nox4, p22phox, and phospho-Smad3 levels normalized to actin levels and presented as fold change compared with vehicle-treated cells for 48 h. Values are mean ± SE. *p < 0.05, **p < 0.01, and ***p < 0.001. TGF-β1, transforming growth factor β1.

Article Snippet: Immunodetection was performed as described previously ( 48 ) using as primary antibody anti-NOX4 (OriGene); anti-pSmad3 (ser 423) (Abcam); anti-Smad3 (Abcam); anti-p22 phox (Santa Cruz Biotechnology, Inc.), anti-NIS (Thermo Scientific); anti-BRAF V600E (Spring); anti-β-actin (Sigma-Aldrich), and anti-Vinculin (Abcam).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Inhibition

Journal: Antioxidants & Redox Signaling

Article Title: NADPH Oxidase NOX4 Is a Critical Mediator of BRAF V600E -Induced Downregulation of the Sodium/Iodide Symporter in Papillary Thyroid Carcinomas

doi: 10.1089/ars.2015.6616

Figure Lengend Snippet: ROS generating NOX4 downregulates the NIS in both BRAFV600E-mutated thyroid cell lines. (A) Expression of NIS (mRNA and protein) 24 h after treatment of BCPAP cells with 1 mM of N-acetyl-cysteine (NAC). Densitometric quantification of NIS protein level normalized to vinculin level and presented as fold change compared with vehicle-treated cells for 24 h. (B) Expression of NIS (mRNA) 24 h after treatment of BCPAP cells with 2 μM of diphenyleneiodonium (DPI). (C) Expression of NIS (mRNA and protein) 48 h after knocking down of NOX4 in BCPAP cells. Densitometric quantification of Nox4 and NIS levels normalized to actin levels and presented as fold change compared with siRNA control-treated cells for 48 h (D) Western blot analysis of cell surface NIS expression in BCPAP cells, 48 h after knocking down of NOX4 or p22phox by RNA interference. Red Ponceau staining at a corresponding molecular weight area of NIS protein was used as loading control. Densitometric quantification of NIS (hyperglycosylated form of 100 kDa) levels normalized to loading control levels and presented as fold change compared with siRNA control-treated cells for 48 h. Values are mean ± SE. *p < 0.05 and **p < 0.01. NIS, sodium/iodide symporter.

Article Snippet: Immunodetection was performed as described previously ( 48 ) using as primary antibody anti-NOX4 (OriGene); anti-pSmad3 (ser 423) (Abcam); anti-Smad3 (Abcam); anti-p22 phox (Santa Cruz Biotechnology, Inc.), anti-NIS (Thermo Scientific); anti-BRAF V600E (Spring); anti-β-actin (Sigma-Aldrich), and anti-Vinculin (Abcam).

Techniques: Expressing, Western Blot, Staining, Molecular Weight

Journal: Antioxidants & Redox Signaling

Article Title: NADPH Oxidase NOX4 Is a Critical Mediator of BRAF V600E -Induced Downregulation of the Sodium/Iodide Symporter in Papillary Thyroid Carcinomas

doi: 10.1089/ars.2015.6616

Figure Lengend Snippet: NOX4 induced by BRAFV600Evia Smad3 signaling pathway downregulates NIS in thyroid PCCL3 cells. (A) Western blot analysis of BRAFV600E and NOX4 protein expressions in PC-BRAFV600E cells treated for 48 h with or without doxycycline (1 μg/ml). Densitometric quantification of BRAFV600E levels normalized to actin levels and presented as fold change compared with vehicle-treated cells for 48 h. Densitometric quantification of Nox4 levels normalized to actin levels and presented as fold change compared with siRNA control-treated cells for 48 h in the absence of doxycycline. (B) QRT-PCR analysis of NIS and NOX4 mRNA levels in PC-BRAFV600E cells treated for 48 h with or without doxycycline (1 μg/ml) and SIS3 (10 μM). (C) qRT-PCR analysis of NIS and NOX4 mRNA levels in PC-BRAFV600E cells treated for 48 h in the absence or presence of TSH (1 mU/l, doxycycline [1 μg/ml] and specific siRNAs [10 nM]). Values are mean ± SE. *p < 0.05, **p < 0.01, and ***p < 0.001. TSH, thyroid stimulating hormone.

Article Snippet: Immunodetection was performed as described previously ( 48 ) using as primary antibody anti-NOX4 (OriGene); anti-pSmad3 (ser 423) (Abcam); anti-Smad3 (Abcam); anti-p22 phox (Santa Cruz Biotechnology, Inc.), anti-NIS (Thermo Scientific); anti-BRAF V600E (Spring); anti-β-actin (Sigma-Aldrich), and anti-Vinculin (Abcam).

Techniques: Western Blot, Quantitative RT-PCR

Journal: Antioxidants & Redox Signaling

Article Title: NADPH Oxidase NOX4 Is a Critical Mediator of BRAF V600E -Induced Downregulation of the Sodium/Iodide Symporter in Papillary Thyroid Carcinomas

doi: 10.1089/ars.2015.6616

Figure Lengend Snippet: NOX4 is involved in TGF-β-induced downregulation of NIS in human thyrocytes. (A) Time course expression of NOX4 and p22phox mRNAs (qRT-PCR) in human thyrocytes treated with human recombinant TGF-β (10 ng/ml). (B) Western blot analysis of NOX4, Smad3, and phospho-Smad3 in human thyrocytes treated for 12 h in the absence or presence of human recombinant TGF-β (10 ng/ml) and SIS3 (10 μM). Densitometric quantification of Nox4 and phospho-Smad3 levels normalized to actin levels and presented as fold change compared with untreated cells. Student t-test is realized by comparing TGF-β with Ctrl and TGF-β+SIS3 with TGF-β. (C) Time course analysis of expression of NIS mRNA (qRT-PCR) in human thyrocytes treated with human recombinant TGF-β (10 ng/ml). (D) qRT-PCR analysis of NIS mRNA expression in human thyrocytes treated or not by human recombinant TGF-β (10 ng/ml for 12 h) in the presence of specific siRNAs (10 nM for 48 h). Values are mean ± SE. *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: Immunodetection was performed as described previously ( 48 ) using as primary antibody anti-NOX4 (OriGene); anti-pSmad3 (ser 423) (Abcam); anti-Smad3 (Abcam); anti-p22 phox (Santa Cruz Biotechnology, Inc.), anti-NIS (Thermo Scientific); anti-BRAF V600E (Spring); anti-β-actin (Sigma-Aldrich), and anti-Vinculin (Abcam).

Techniques: Expressing, Quantitative RT-PCR, Recombinant, Western Blot

Journal: Antioxidants & Redox Signaling

Article Title: NADPH Oxidase NOX4 Is a Critical Mediator of BRAF V600E -Induced Downregulation of the Sodium/Iodide Symporter in Papillary Thyroid Carcinomas

doi: 10.1089/ars.2015.6616

Figure Lengend Snippet: High level of NOX4 mRNA is correlated with low level of NIS mRNA and low TDS in papillary thyroid cancer (PTC) harboring BRAFV600E mutation. A homogeneous cohort of 390 PTCs (170 BRAFWT PTCs and 220 BRAFV600E PTCs) were included in this analysis. Correlative analysis between NOX4 mRNA (A) or NIS mRNA levels (B) and BRAFV600E mutation in 390 PTCs. Correlation of NOX4 mRNA level and NIS mRNA level in 220 PTCs harboring BRAFV600E mutation (C). Correlative analysis between BRAFV600E mutation and TDS in 390 PTCs (D). Correlative analysis between TGF-β1 mRNA (E) or Smad3 mRNA levels (F) and BRAFV600E mutation in 390 PTCs. TDS, Thyroid Differentiation Score. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars

Article Snippet: Immunodetection was performed as described previously ( 48 ) using as primary antibody anti-NOX4 (OriGene); anti-pSmad3 (ser 423) (Abcam); anti-Smad3 (Abcam); anti-p22 phox (Santa Cruz Biotechnology, Inc.), anti-NIS (Thermo Scientific); anti-BRAF V600E (Spring); anti-β-actin (Sigma-Aldrich), and anti-Vinculin (Abcam).

Techniques: Mutagenesis

Journal: Antioxidants & Redox Signaling

Article Title: NADPH Oxidase NOX4 Is a Critical Mediator of BRAF V600E -Induced Downregulation of the Sodium/Iodide Symporter in Papillary Thyroid Carcinomas

doi: 10.1089/ars.2015.6616

Figure Lengend Snippet: Oncogenic BRAF-dependent regulation of NOX4. (A) A schematic model of all the steps involved in the oncogenic BRAF-dependent regulation of NOX4. Our data show that BRAFV600E upregulates NOX4 expression via the TGF-B1/Smad3 signaling pathway in thyroid cancer cells. The NOX4-dependent ROS production plays a key role in SLC5A5 gene repression. Consequently, it might be involved in the absence of radioiodide uptake by BRAF-mutated cancer cells. The targets of different inhibitors used in this study are indicated. (B) Ontogenic changes of mRNA expression of NIS, TPO, and NOX4. qRT-PCR analysis of NOX4, TPO, and NIS mRNA expression in 6 human thyroids at 8, 10, or 12 weeks of development. GW, gestational week; TPO, thyroperoxydase.

Article Snippet: Immunodetection was performed as described previously ( 48 ) using as primary antibody anti-NOX4 (OriGene); anti-pSmad3 (ser 423) (Abcam); anti-Smad3 (Abcam); anti-p22 phox (Santa Cruz Biotechnology, Inc.), anti-NIS (Thermo Scientific); anti-BRAF V600E (Spring); anti-β-actin (Sigma-Aldrich), and anti-Vinculin (Abcam).

Techniques: Expressing, Quantitative RT-PCR

Journal: Antioxidants & Redox Signaling

Article Title: NADPH Oxidase NOX4 Is a Critical Mediator of BRAF V600E -Induced Downregulation of the Sodium/Iodide Symporter in Papillary Thyroid Carcinomas

doi: 10.1089/ars.2015.6616

Figure Lengend Snippet: High level of NOX4 mRNA is correlated with low thyroid differentiation score in genetically engineered mouse models of thyroid cancer. Gene expression profiling of GEM models of thyroid hyperplasia or the indicated thyroid cancer phenotypes. (A) Fold change in NOX4 expression compared to normal thyroid controls calculated from Affymetrix expression array data. (B) Association of NOX4 expression levels with the TGF-β transcriptional output score. Pearson correlation was used to calculate r- and p-values. (C) Thyroid differentiation score for each mouse model. (D) Correlation between thyroid differentiation score and NOX4 expression. Pearson correlation was used to calculate r- and p-values.

Article Snippet: Immunodetection was performed as described previously ( 48 ) using as primary antibody anti-NOX4 (OriGene); anti-pSmad3 (ser 423) (Abcam); anti-Smad3 (Abcam); anti-p22 phox (Santa Cruz Biotechnology, Inc.), anti-NIS (Thermo Scientific); anti-BRAF V600E (Spring); anti-β-actin (Sigma-Aldrich), and anti-Vinculin (Abcam).

Techniques: Expressing