Journal: Indian Journal of Pharmacology

Article Title: Therapeutic potential of diosmin, a citrus flavonoid against arsenic-induced neurotoxicity via suppression of NOX 4 and its subunits

doi: 10.4103/ijp.IJP_837_19

Figure Lengend Snippet: Diosmin treatment effect on P47 phox expression (a) normal control (b) arsenic control group (c) apocynin 10 mg/kg (d) diosmin (50 mg/kg) and (e) diosmin (100 mg/kg)

Article Snippet: Sodium arsenite from Hi media chemicals (Mumbai, India) and antibodies (NOX4, P47 phox and gp91 phox ) were procured from St. John's laboratory Ltd., London.

Techniques: Expressing, Control

Journal: Indian Journal of Pharmacology

Article Title: Therapeutic potential of diosmin, a citrus flavonoid against arsenic-induced neurotoxicity via suppression of NOX 4 and its subunits

doi: 10.4103/ijp.IJP_837_19

Figure Lengend Snippet: Integrated densities of NOX 4, gp91 phox , P47 phox expressions (a) indicating the expression of NOX 4 in different treatment groups (b) the expression of gp91 phox in different treatment groups (c) the expression of P47 phox in different treatment groups. * P < 0.05 is considered significant in comparison with normal and # P < 0.05 is considered significant in comparison with arsenic control

Article Snippet: Sodium arsenite from Hi media chemicals (Mumbai, India) and antibodies (NOX4, P47 phox and gp91 phox ) were procured from St. John's laboratory Ltd., London.

Techniques: Expressing, Comparison, Control

Journal: Biomedicines

Article Title: Secretome of hESC-Derived MSC-like Immune and Matrix Regulatory Cells Mitigate Pulmonary Fibrosis through Antioxidant and Anti-Inflammatory Effects

doi: 10.3390/biomedicines11020463

Figure Lengend Snippet: Elevated Nox-mediated oxidative stress and Tlr4/MyD88-mediated inflammation in lungs of mice with BLM-induced pulmonary fibrosis (PF). ( a ) Schematic diagram for the creation of the BLM-induced PF mouse model. C57BL/6 mice that were 6–8 weeks old were injured by intratracheal instillation of 1.5 mg/kg BLM at day 0 (d0) and d7, and the lung tissues were harvested at d14 post-injury for evaluation (N = 8). ( b ) Representative histochemical images of H&E and Masson staining demonstrated alveolar structural damage (H&E staining) with ECM deposition (Masson staining). ( c ) Representative immunoblotting blots demonstrated an increased expression of proteins of Nox family numbers (Nox2, Nox3, and Nox4), oxidization stress markers (Nrf2 and Ho-1), and EMT markers (α-SMA and Vimentin) in the lungs of BLM-induced PF mice compared with the saline group. ( d ) Semi-quantitation of relative levels of proteins of interest as determined by the relative density over Gapdh in ( c ). ( e ) IF staining validated the increased abundance of α-SMA in the lungs of mice at d14 post-BLM challenge compared with saline controls. ( f ) Representative blots of immunoblotting assay demonstrated the enhanced activation of Tlr4/MyD88 signaling in the lung of BLM-induced PF mice model compared with the saline group. ( g ) Semi-quantitation of the relative levels of proteins of interest Tlr4, MyD88, and p-NF-κB as evaluated by the relative density over Gapdh in ( f ). Data in ( d , g ) represent mean ± SD from the indicated number of mice (N = 3), as analyzed by GraphPad prism one-way ANOVA Tukey’s multiple comparisons test.

Article Snippet: After blocking with 5% non-fat milk or 3% BSA in PBS for 1 h at RT, the membrane was incubated overnight at 4 °C with primary antibodies: rabbit monoclonal IgG HO-1 (27282-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG NRF2 (Proteintech, 16396-1-AP), rabbit monoclonal IgG SMA (14395-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG NOX4 (BA2813, Boster, Wuhan, China), mouse monoclonal IgG NOX2 (BA2811, Boster, Wuhan, China), mouse monoclonal IgG anti-α-tubulin (T5168, SIGMA, St. Louis, MO, USA), rabbit monoclonal IgG ATF6 (24169-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG N-cadherin (22018-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG NOX3 (20065-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG Toll-like receptor 4 (TLR4) (BS20594R, Bioss, Woburn, MA, USA), and rabbit monoclonal IgG Myeloid differentiation primary response 88 (MyD88) (NB100-56698, Novusbio, Centennial, CO, USA).

Techniques: Staining, Western Blot, Expressing, Saline, Quantitation Assay, Activation Assay

Journal: Biomedicines

Article Title: Secretome of hESC-Derived MSC-like Immune and Matrix Regulatory Cells Mitigate Pulmonary Fibrosis through Antioxidant and Anti-Inflammatory Effects

doi: 10.3390/biomedicines11020463

Figure Lengend Snippet: BLM induces NOX4-mediated oxidative stress in A549 lung epithelial cells. ( a ) Representative images of immunoblotting blots revealed an increased expression of α-SMA, NOX4 and NRF2 in A549 cells exposed to indicated concentrations of BLM (1.0, 2.5, 5.0, 10.0, and 20.0 μg/mL) for 48 h. ( b ) Semi-quantitation of the relative levels of α-SMA, NOX4, and NRF2 proteins as evaluated by the relative density over GAPDH in ( a ). ( c ) Representative images of A549 cells treated with indicated concentrations of BLM for 48 h and stained with CellROX ® Orange reagent. The BLM-induced A549 injury increased ROS production, especially at the concentration of 5 μg/mL, compared to untreated cells (NC). ( d ) Mean gray value of ROS production in ( c ). ( e ) Representative images of IF exhibited more abundant HO-1, NRF2, NOX4, ATF6, α-SMA, and N-Cad proteins in A549 cells exposed to 5 μg/mL of BLM for 48 h as compared with the NC control. ( f ) Representative images of the fluorescent intensity of the relevant proteins in ( e ). Data in ( b , d , f ) represent mean ± SD from three independent experiments (N = 3), as analyzed by GraphPad prism one-way ANOVA Tukey’s multiple comparisons test. Scale bars in ( c ), 100 µm; ( e ), 10 µm.

Article Snippet: After blocking with 5% non-fat milk or 3% BSA in PBS for 1 h at RT, the membrane was incubated overnight at 4 °C with primary antibodies: rabbit monoclonal IgG HO-1 (27282-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG NRF2 (Proteintech, 16396-1-AP), rabbit monoclonal IgG SMA (14395-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG NOX4 (BA2813, Boster, Wuhan, China), mouse monoclonal IgG NOX2 (BA2811, Boster, Wuhan, China), mouse monoclonal IgG anti-α-tubulin (T5168, SIGMA, St. Louis, MO, USA), rabbit monoclonal IgG ATF6 (24169-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG N-cadherin (22018-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG NOX3 (20065-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG Toll-like receptor 4 (TLR4) (BS20594R, Bioss, Woburn, MA, USA), and rabbit monoclonal IgG Myeloid differentiation primary response 88 (MyD88) (NB100-56698, Novusbio, Centennial, CO, USA).

Techniques: Western Blot, Expressing, Quantitation Assay, Staining, Concentration Assay, Control

Journal: Biomedicines

Article Title: Secretome of hESC-Derived MSC-like Immune and Matrix Regulatory Cells Mitigate Pulmonary Fibrosis through Antioxidant and Anti-Inflammatory Effects

doi: 10.3390/biomedicines11020463

Figure Lengend Snippet: hESC-MSC-IMRC-CM reduces the BLM-induced oxidative stress and EMT in A549 lung epithelial cells. ( a ) Representative immunoblots showed that hESC-MSC-IMRC-CM could reduce BLM-induced A549 cells oxidative injury by decreasing the expression of NRF2, HO-1, NOX4, and EMT markers’ N-Cad. ( b ) The relative levels of NRF2, HO-1, NOX4, and N-Cad proteins as evaluated by a densitometric analysis in ( a ). ( c ) Representative images of A549 cells treated with indicated conditions for 48 h and stained with CellROX ® Orange reagent. hESC-MSC-IMRC-CM significantly reduced the ROS production compared to the BLM group. ( d ) Mean gray value of ROS production in ( c ). ( e ) hESC-MSC-IMRC-CM increased glutathione peroxidase (GSH-PX) production in A549 cells compared to untreated controls. Data in ( b , d , e ) represent mean ± SD of three independent experiments (N = 3), as analyzed by GraphPad prism Two-way ANOVA Tukey’s multiple comparisons test. Scale bars in ( c ), 100 μm.

Article Snippet: After blocking with 5% non-fat milk or 3% BSA in PBS for 1 h at RT, the membrane was incubated overnight at 4 °C with primary antibodies: rabbit monoclonal IgG HO-1 (27282-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG NRF2 (Proteintech, 16396-1-AP), rabbit monoclonal IgG SMA (14395-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG NOX4 (BA2813, Boster, Wuhan, China), mouse monoclonal IgG NOX2 (BA2811, Boster, Wuhan, China), mouse monoclonal IgG anti-α-tubulin (T5168, SIGMA, St. Louis, MO, USA), rabbit monoclonal IgG ATF6 (24169-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG N-cadherin (22018-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG NOX3 (20065-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG Toll-like receptor 4 (TLR4) (BS20594R, Bioss, Woburn, MA, USA), and rabbit monoclonal IgG Myeloid differentiation primary response 88 (MyD88) (NB100-56698, Novusbio, Centennial, CO, USA).

Techniques: Western Blot, Expressing, Staining

Journal: Biomedicines

Article Title: Secretome of hESC-Derived MSC-like Immune and Matrix Regulatory Cells Mitigate Pulmonary Fibrosis through Antioxidant and Anti-Inflammatory Effects

doi: 10.3390/biomedicines11020463

Figure Lengend Snippet: hESC-MSC-IMRCs and hESC-MSC-IMRC-CM inhibit the development of pulmonary fibrosis in mice. ( a ) Schematic illustration shows the experimental workflow for the treatment of the BLM-induced IPF mouse model at an early stage of PF development. C57BL/6 mice were challenged with BLM via the laryngotracheal route at day ( d ) 0 and d7. A 200 μL sample of saline containing 3 × 10 6 cells of hESC-MSC-IMRCs and 200 μL of hESC-MSC-IMRC-CM were delivered via the tail vein injection at d3, d7, and d14 after the first dose of BLM challenge. The lung tissues were harvested at d28 for analysis. ( b ) The effect of hESC-MSC-IMRC-CM on lung index (ratio of lung/body weights). ( c ) Representative histochemical images of H&E and Masson staining demonstrate that both hESC-MSC-IMRC-CM and hESC-MSC-IMRCs can ameliorate the pathogenesis of pulmonary fibrosis (H&E staining) with ECM deposition (Masson staining) compared to animals challenged with BLM alone. ( d ) Histological analysis of the fibrotic area. ( e , f ) The levels of hydroxyproline (Hyp) and malondialdehyde (MDA) contents. ( g ) Representative immunoblots show a reduction in indicated molecules of oxidative stress-signaling pathways and α-SMA in BLM-induced fibrotic lung administrated with hESC-MSC-IMRC-CM or hESC-MSC-IMRCs compared to the lungs of BLM-challenged mice alone. ( h ) The relative levels of Nox4, Nrf2, Ho-1 and α-SMA proteins as evaluated by a densitometric analysis in ( g ). Data in ( b , d – f , h ) represent mean ± SD of three independent experiments (N = 3), as analyzed by GraphPad prism two-way ANOVA Tukey’s multiple comparisons test. Scale bars in ( c ), 200 µm in the first and third panels, 20 µm in the second and fourth rows.

Article Snippet: After blocking with 5% non-fat milk or 3% BSA in PBS for 1 h at RT, the membrane was incubated overnight at 4 °C with primary antibodies: rabbit monoclonal IgG HO-1 (27282-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG NRF2 (Proteintech, 16396-1-AP), rabbit monoclonal IgG SMA (14395-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG NOX4 (BA2813, Boster, Wuhan, China), mouse monoclonal IgG NOX2 (BA2811, Boster, Wuhan, China), mouse monoclonal IgG anti-α-tubulin (T5168, SIGMA, St. Louis, MO, USA), rabbit monoclonal IgG ATF6 (24169-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG N-cadherin (22018-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG NOX3 (20065-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG Toll-like receptor 4 (TLR4) (BS20594R, Bioss, Woburn, MA, USA), and rabbit monoclonal IgG Myeloid differentiation primary response 88 (MyD88) (NB100-56698, Novusbio, Centennial, CO, USA).

Techniques: Saline, Injection, Staining, Western Blot, Protein-Protein interactions

Journal: Biomedicines

Article Title: Secretome of hESC-Derived MSC-like Immune and Matrix Regulatory Cells Mitigate Pulmonary Fibrosis through Antioxidant and Anti-Inflammatory Effects

doi: 10.3390/biomedicines11020463

Figure Lengend Snippet: hESC-MSC-IMRC-CM alleviates the progression of pulmonary fibrosis in mice. ( a ) Schematic illustration shows the experimental workflow for treatments of late-stage of PF disease in the BLM-induced PF mouse model. C57BL/6 mice were challenged with BLM via the laryngotracheal route at day ( d ) 0 and d7. A 200 μL sample of saline containing 3 × 10 6 cells of hESC-MSC-IMRCs or 200 μL hESC-MSC-IMRC-CM was administrated via tail vein injection at d7, d14, and d21 after the first dose of BLM challenge. The lung tissues were harvested at d28 for analysis. ( b ) The ratio of lung weight/body weight (LW/BW). ( c ) Representative histochemical images of H&E and Masson staining demonstrate that both hESC-MSC-IMRC-CM and hESC-MSC-IMRCs can ameliorate the progression of pulmonary fibrosis (H&E staining) with ECM deposition (Masson staining) compared to the BLM-group. ( d ) Histological analysis of the fibrotic area. ( e , f ) The level of hydroxyproline (Hyp) and malondialdehyde (MDA) contents. ( g ) Representative immunoblots show a decreased expression of indicated components of oxidative stress signaling cascade and α-SMA in BLM-induced lung treated with hESC-MSC-IMRC-CM or hESC-MSC-IMRCs compared to the untreated BLM group. ( h ) The relative levels of Nox4, Nrf2, Ho-1, and α-SMA proteins as evaluated by a densitometric analysis in ( g ). Data in ( b , d – f , h ) represent mean ± SD of three independent experiments (N = 3), as analyzed by GraphPad prism two-way ANOVA Tukey’s multiple comparisons test. Scale bars in ( c ), 200 µm in the first and third rows, 20 µm in the second and fourth rows.

Article Snippet: After blocking with 5% non-fat milk or 3% BSA in PBS for 1 h at RT, the membrane was incubated overnight at 4 °C with primary antibodies: rabbit monoclonal IgG HO-1 (27282-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG NRF2 (Proteintech, 16396-1-AP), rabbit monoclonal IgG SMA (14395-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG NOX4 (BA2813, Boster, Wuhan, China), mouse monoclonal IgG NOX2 (BA2811, Boster, Wuhan, China), mouse monoclonal IgG anti-α-tubulin (T5168, SIGMA, St. Louis, MO, USA), rabbit monoclonal IgG ATF6 (24169-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG N-cadherin (22018-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG NOX3 (20065-1-AP, Proteintech, Rosemont, IL, USA), rabbit monoclonal IgG Toll-like receptor 4 (TLR4) (BS20594R, Bioss, Woburn, MA, USA), and rabbit monoclonal IgG Myeloid differentiation primary response 88 (MyD88) (NB100-56698, Novusbio, Centennial, CO, USA).

Techniques: Saline, Injection, Staining, Western Blot, Expressing

Journal: Antioxidants

Article Title: Role of NADPH Oxidase 4 in Corneal Endothelial Cells Is Mediated by Endoplasmic Reticulum Stress and Autophagy

doi: 10.3390/antiox12061228

Figure Lengend Snippet: The in vivo transfection of genes. ( A , B ) The transfection efficiency using electroporation was evaluated with pGFP and fluorescein-conjugated siRNA. ( C , D ) The levels of NOX4 were evaluated using the immunofluorescence staining of NOX4 at one week after transfection. Scale bar = 150 µm. ( E , F ) The levels of 8-OHdG were evaluated using immunofluorescence staining of 8-OHdG at one week after transfection. Scale bar = 100 µm. ( G ) The in vivo staining of JC-1 and ER-Tracker red was performed to evaluate the mitochondrial membrane potential and ER stress. Scale bar = 100 µm * p < 0.05, ** p < 0.01.

Article Snippet: The samples were incubated overnight with mouse anti-NOX4 antibody (MABC616, Merck Millipore, Burlington, MA, USA), mouse anti-8-OHdG antibody (ab62623, Abcam, Cambridge, UK), rabbit anti-human NF-κB antibody (sc-372; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or rabbit anti-X-box binding protein 1 (XBP-1) antibody (sc-7160, Santa Cruz Biotechnology) at 4 °C.

Techniques: In Vivo, Transfection, Electroporation, Immunofluorescence, Staining

Journal: Antioxidants

Article Title: Role of NADPH Oxidase 4 in Corneal Endothelial Cells Is Mediated by Endoplasmic Reticulum Stress and Autophagy

doi: 10.3390/antiox12061228

Figure Lengend Snippet: NOX4 regulates the cell death of corneal-endothelial cells in rat. ( A – C ) The corneas were evaluated after transfections of siNOX4 or pNOX4. The corneal opacity was graded. ( D , E ) Alizarin S red staining showed the corneal-endothelial cell density at one week and two weeks. * p < 0.05.

Article Snippet: The samples were incubated overnight with mouse anti-NOX4 antibody (MABC616, Merck Millipore, Burlington, MA, USA), mouse anti-8-OHdG antibody (ab62623, Abcam, Cambridge, UK), rabbit anti-human NF-κB antibody (sc-372; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or rabbit anti-X-box binding protein 1 (XBP-1) antibody (sc-7160, Santa Cruz Biotechnology) at 4 °C.

Techniques: Transfection, Staining

Journal: Antioxidants

Article Title: Role of NADPH Oxidase 4 in Corneal Endothelial Cells Is Mediated by Endoplasmic Reticulum Stress and Autophagy

doi: 10.3390/antiox12061228

Figure Lengend Snippet: The effect of NOX4 on cryo-injured cornea. ( A – C ) Corneas were injured with cryoinjury after transfections of siNOX4 or pNOX4. Corneal opacity was evaluated. ( D , E ) Corneal-endothelial cells stained with Alizarin S red solution showed a recovery rate. * p < 0.05.

Article Snippet: The samples were incubated overnight with mouse anti-NOX4 antibody (MABC616, Merck Millipore, Burlington, MA, USA), mouse anti-8-OHdG antibody (ab62623, Abcam, Cambridge, UK), rabbit anti-human NF-κB antibody (sc-372; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or rabbit anti-X-box binding protein 1 (XBP-1) antibody (sc-7160, Santa Cruz Biotechnology) at 4 °C.

Techniques: Transfection, Staining

Journal: Antioxidants

Article Title: Role of NADPH Oxidase 4 in Corneal Endothelial Cells Is Mediated by Endoplasmic Reticulum Stress and Autophagy

doi: 10.3390/antiox12061228

Figure Lengend Snippet: NOX4 regulates the cell shape and cell proliferation. ( A ) NOX4 mRNA expressions were evaluated after the transfection of siNOX4 or pNOX4 at 72 h after treatment. ( B , C ) The cell shape was observed using an inverted microscopy. The cell shape was evaluated using the elongation factor. Data presented as mean ± S.D. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Article Snippet: The samples were incubated overnight with mouse anti-NOX4 antibody (MABC616, Merck Millipore, Burlington, MA, USA), mouse anti-8-OHdG antibody (ab62623, Abcam, Cambridge, UK), rabbit anti-human NF-κB antibody (sc-372; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or rabbit anti-X-box binding protein 1 (XBP-1) antibody (sc-7160, Santa Cruz Biotechnology) at 4 °C.

Techniques: Transfection, Inverted Microscopy

Journal: Antioxidants

Article Title: Role of NADPH Oxidase 4 in Corneal Endothelial Cells Is Mediated by Endoplasmic Reticulum Stress and Autophagy

doi: 10.3390/antiox12061228

Figure Lengend Snippet: NOX4 regulates senescence. ( A , B ) A senescence-associated β-galactosidase assay was performed as a senescence marker at 72 h after treatment. ( C ) Cell viability was measured using the CCK-8 kit. ( D ) The cell proliferation rate was measured by a BrdU incorporation assay. ( E – G ) Mitochondrial oxidative stress levels were measured using a MitoSOX probe. Scale bar = 100 µm. Data presented as mean ± S.D. * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Article Snippet: The samples were incubated overnight with mouse anti-NOX4 antibody (MABC616, Merck Millipore, Burlington, MA, USA), mouse anti-8-OHdG antibody (ab62623, Abcam, Cambridge, UK), rabbit anti-human NF-κB antibody (sc-372; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or rabbit anti-X-box binding protein 1 (XBP-1) antibody (sc-7160, Santa Cruz Biotechnology) at 4 °C.

Techniques: Marker, CCK-8 Assay, BrdU Incorporation Assay

Journal: Antioxidants

Article Title: Role of NADPH Oxidase 4 in Corneal Endothelial Cells Is Mediated by Endoplasmic Reticulum Stress and Autophagy

doi: 10.3390/antiox12061228

Figure Lengend Snippet: NOX4 regulates the ATF4 and XBP-1 branches of ER stress. ( A ) ER organelle visualization by ER-Tracker TM red fluorescence imaging at 72 h after treatment. Scale bar = 100 µm. ( B , C ) ATF4 levels were evaluated by Western blotting, after transfections of siRNA for NOX4, or plasmids for NOX4. ( D , E ) RT-qPCR and Western blotting analyses for ATF6 were performed. ( F , G ) Immunofluorescence staining of XBP-1 was performed to evaluate the XBP-1 nuclear translocation. ( H ) Western blotting of XBP-1 was performed to evaluate the translocation of XBP-1. Data presented as mean ± S.D. * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: The samples were incubated overnight with mouse anti-NOX4 antibody (MABC616, Merck Millipore, Burlington, MA, USA), mouse anti-8-OHdG antibody (ab62623, Abcam, Cambridge, UK), rabbit anti-human NF-κB antibody (sc-372; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or rabbit anti-X-box binding protein 1 (XBP-1) antibody (sc-7160, Santa Cruz Biotechnology) at 4 °C.

Techniques: Fluorescence, Imaging, Western Blot, Transfection, Quantitative RT-PCR, Immunofluorescence, Staining, Translocation Assay

Journal: Antioxidants

Article Title: Role of NADPH Oxidase 4 in Corneal Endothelial Cells Is Mediated by Endoplasmic Reticulum Stress and Autophagy

doi: 10.3390/antiox12061228

Figure Lengend Snippet: NOX4 regulates autophagy through the mitochondria. ( A , B ) The mitochondrial membrane potential was measured using MitoPotential kit at 72 h after treatment. ( C ) The JC-1 probe was used for obtaining images of the mitochondrial membrane potential. ( D ) Lysosome was visualized by LysoTracker green, and mitochondria was visualized by MitoTracker red. ( E , F ) Autophagy was evaluated by the Western blot of LC3. Data presented as mean ± S.D. * p < 0.05, ** p < 0.01 and *** p < 0.001.

Article Snippet: The samples were incubated overnight with mouse anti-NOX4 antibody (MABC616, Merck Millipore, Burlington, MA, USA), mouse anti-8-OHdG antibody (ab62623, Abcam, Cambridge, UK), rabbit anti-human NF-κB antibody (sc-372; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or rabbit anti-X-box binding protein 1 (XBP-1) antibody (sc-7160, Santa Cruz Biotechnology) at 4 °C.

Techniques: Western Blot

Journal: Frontiers in Pharmacology

Article Title: Interaction Mechanisms Between the NOX4/ROS and RhoA/ROCK1 Signaling Pathways as New Anti- fibrosis Targets of Ursolic Acid in Hepatic Stellate Cells

doi: 10.3389/fphar.2019.00431

Figure Lengend Snippet: Primer sequences for RT-qPCR.

Article Snippet: The full-length human recombinant NOX4 (cat #: H00050507-G01) and RhoA (cat #: H00000387-P01) proteins were purchased from Abnova (Walnut, CA, United States).

Techniques: Sequencing

Journal: Frontiers in Pharmacology

Article Title: Interaction Mechanisms Between the NOX4/ROS and RhoA/ROCK1 Signaling Pathways as New Anti- fibrosis Targets of Ursolic Acid in Hepatic Stellate Cells

doi: 10.3389/fphar.2019.00431

Figure Lengend Snippet: Construction and identification of stable cell lines. (A) The HSC-T6 cell line was transfected with a lentivirus containing NOX4 shRNA. Cells were observed under an optical microscope (left) and a fluorescence (GFP) microscope (right) in the same field of vision. (B) RT-qPCR analysis of Nox4 mRNA in the NOX4 shRNA stable cell line after TGF-β1 treatment (10 ng/mL) for 48 h. (C) WB analysis of the NOX4 protein levels in the NOX4i group. (D) The HSC-T6 cell line was transfected with a lentivirus containing RhoA shRNA. Cells were observed under an optical microscope (left) and a fluorescence microscope (right) in the same field of vision. (E) RT-qPCR analysis of Rhoa mRNA in the Rhoa shRNA stable cell line. (F) WB analysis of the RhoA protein levels in the RhoAi group. (G) The HSC-T6 cell line was transfected with a Nox4 overexpression lentivirus. Cells were observed under an optical microscope (left) and a fluorescence microscope (right) in the same field of vision. (H) RT-qPCR analysis of Nox4 mRNA in the NOX4 overexpression stable cell line. (I) WB analysis of the NOX4 protein levels in the NOX4 GV 358Lv group. Original magnification, 200×. The data are presented as the means ± SEMs of three replicates. ∗ p < 0.05 versus the CON group (lentivirus containing a scramble sequence).

Article Snippet: The full-length human recombinant NOX4 (cat #: H00050507-G01) and RhoA (cat #: H00000387-P01) proteins were purchased from Abnova (Walnut, CA, United States).

Techniques: Stable Transfection, Transfection, shRNA, Microscopy, Fluorescence, Quantitative RT-PCR, Over Expression, Sequencing

Journal: Frontiers in Pharmacology

Article Title: Interaction Mechanisms Between the NOX4/ROS and RhoA/ROCK1 Signaling Pathways as New Anti- fibrosis Targets of Ursolic Acid in Hepatic Stellate Cells

doi: 10.3389/fphar.2019.00431

Figure Lengend Snippet: The regulatory effects of NOX4 and RhoA as well as the UA intervention effects on the migration and invasion of HSCs after TGF-β1 treatment (10 ng/mL) for 48 h. (A) Cell proliferation detected by the MTS assay. (B,C) Cell apoptosis levels detected by the TUNEL assay. Black arrows: typical apoptotic cells. Original magnification, 200×. Percentage of apoptotic cells = apoptotic cells/cell count per high-power field; 10 different fields of vision were randomly selected in each group. (D,E) Cell scratch assay. Original magnification, 100×. (F,H) Cell invasion detected by the Transwell invasion system. Original magnification, 200×; (G) MTS indirect cell count curve. r = 0.9959. The data are presented as the means ± SEMs of three replicates. ∗ p < 0.05, ∗∗ p < 0.01 versus the CON group; # p < 0.05, ## p < 0.01 versus the NOX4 GV 358Lv group. ∆ p < 0.05 versus the NOX4i group.

Article Snippet: The full-length human recombinant NOX4 (cat #: H00050507-G01) and RhoA (cat #: H00000387-P01) proteins were purchased from Abnova (Walnut, CA, United States).

Techniques: Migration, MTS Assay, TUNEL Assay, Cell Counting, Wound Healing Assay

Journal: Frontiers in Pharmacology

Article Title: Interaction Mechanisms Between the NOX4/ROS and RhoA/ROCK1 Signaling Pathways as New Anti- fibrosis Targets of Ursolic Acid in Hepatic Stellate Cells

doi: 10.3389/fphar.2019.00431

Figure Lengend Snippet: The regulatory effects of NOX4 and RhoA as well as the UA intervention effects on the F-actin polymerization levels in each group after TGF-β1 treatment (10 ng/mL) for 48 h. (A,B) WB analysis of F-actin in each group. The data are presented as the means ± SEMs of three replicates. ∗ p < 0.05, ∗∗ p < 0.01 versus the CON group; # p < 0.05, ## p < 0.01 versus the NOX4 GV 358Lv group. (C,D) F-actin immunofluorescence staining in each group. Mean integral optical density (IOD) values were measured by Image Pro Plus 6.0 software; 10 different fields of vision were randomly selected. Original magnification, 400×.

Article Snippet: The full-length human recombinant NOX4 (cat #: H00050507-G01) and RhoA (cat #: H00000387-P01) proteins were purchased from Abnova (Walnut, CA, United States).

Techniques: Immunofluorescence, Staining, Software

Journal: Frontiers in Pharmacology

Article Title: Interaction Mechanisms Between the NOX4/ROS and RhoA/ROCK1 Signaling Pathways as New Anti- fibrosis Targets of Ursolic Acid in Hepatic Stellate Cells

doi: 10.3389/fphar.2019.00431

Figure Lengend Snippet: UA intervention effects on the mRNA and protein expression of the NOX4/ROS and RhoA/ROCK1 signaling pathways. (A,B) RT-qPCR analysis of Nox4, Rhoa, Rock1, Alphasma, Col1a1, mmp1, and Timp1 mRNA expression levels in each group. (C–E) WB analysis of the protein expression levels in each group. (F) ROS contents in each group detected by the DCFH-DA assay. The data are presented as the means ± SEMs of three replicates. ∗ p < 0.05, versus the CON group; # p < 0.05, versus the NOX4 GV 358Lv group.

Article Snippet: The full-length human recombinant NOX4 (cat #: H00050507-G01) and RhoA (cat #: H00000387-P01) proteins were purchased from Abnova (Walnut, CA, United States).

Techniques: Expressing, Protein-Protein interactions, Quantitative RT-PCR, DCFH-DA Assay

Journal: Frontiers in Pharmacology

Article Title: Interaction Mechanisms Between the NOX4/ROS and RhoA/ROCK1 Signaling Pathways as New Anti- fibrosis Targets of Ursolic Acid in Hepatic Stellate Cells

doi: 10.3389/fphar.2019.00431

Figure Lengend Snippet: Construction and identification of NOX4 GV 358Lv -RhoA shRNA stable cell lines. (A) The NOX4 GV 358Lv cell line was transfected with a lentivirus containing RhoA shRNA. Cells were observed under an optical microscope (left) and a fluorescence microscope (right) in the same field of vision. Original magnification, 200×; (B) RT-qPCR analysis of Nox4 mRNA in the NOX4 shRNA stable cell line. (C) WB analysis of the NOX4 protein levels in the NOX4 GV 358Lv -RhoAi group. (D) Cell proliferation detected by the MTS assay. (E,F) Cell apoptosis levels detected by the TUNEL assay. Black arrows: typical apoptotic cells. Percentage of apoptotic cells = apoptotic cells/cell count per high-power field; 10 different fields of vision were randomly selected from each group. Original magnification, 200×; (G,H) Cell scratch assay. Original magnification, 100×. The data are presented as the means ± SEMs of three replicates. ∗ p < 0.05, versus the NOX4 GV 358Lv -con group; # p < 0.05, versus the NOX4 GV 358Lv -RhoAi group.

Article Snippet: The full-length human recombinant NOX4 (cat #: H00050507-G01) and RhoA (cat #: H00000387-P01) proteins were purchased from Abnova (Walnut, CA, United States).

Techniques: shRNA, Stable Transfection, Transfection, Microscopy, Fluorescence, Quantitative RT-PCR, MTS Assay, TUNEL Assay, Cell Counting, Wound Healing Assay

Journal: Frontiers in Pharmacology

Article Title: Interaction Mechanisms Between the NOX4/ROS and RhoA/ROCK1 Signaling Pathways as New Anti- fibrosis Targets of Ursolic Acid in Hepatic Stellate Cells

doi: 10.3389/fphar.2019.00431

Figure Lengend Snippet: The invasion levels and F-actin polymerization in the NOX4 GV 358Lv -RhoA shRNA stable cell line. (A,B) Cell invasion detected by the Transwell invasion system. Original magnification, 200×. (C,D) F-actin immunofluorescence staining in each group. Mean IOD values were measured by Image Pro Plus 6.0 software; 10 different fields of visions were selected. Original magnification, 400×. (E,F) WB analysis of F-actin in each group. The data are presented as the means ± SEMs of three replicates. ∗ p < 0.05, versus the NOX4 GV 358Lv -con group; # p < 0.05, versus the NOX4 GV 358Lv -RhoAi group.

Article Snippet: The full-length human recombinant NOX4 (cat #: H00050507-G01) and RhoA (cat #: H00000387-P01) proteins were purchased from Abnova (Walnut, CA, United States).

Techniques: shRNA, Stable Transfection, Immunofluorescence, Staining, Software

Journal: Frontiers in Pharmacology

Article Title: Interaction Mechanisms Between the NOX4/ROS and RhoA/ROCK1 Signaling Pathways as New Anti- fibrosis Targets of Ursolic Acid in Hepatic Stellate Cells

doi: 10.3389/fphar.2019.00431

Figure Lengend Snippet: mRNA and protein expression changes in the NOX4 GV 358Lv -RhoA shRNA stable cell line and direct binding analysis of NOX4 and RhoA. (A) RT-qPCR analysis of the mRNA expression levels in each group. (B,C) WB analysis of the protein expression levels in each group. (D) ROS contents in each group detected by the DCFH-DA assay. The data are presented as the means ± SEMs of three replicates. ∗ p < 0.05, versus the CON group; # p < 0.05, versus the NOX4 GV 358Lv group. (E) Recapitulative overview scheme of the study.

Article Snippet: The full-length human recombinant NOX4 (cat #: H00050507-G01) and RhoA (cat #: H00000387-P01) proteins were purchased from Abnova (Walnut, CA, United States).

Techniques: Expressing, shRNA, Stable Transfection, Binding Assay, Quantitative RT-PCR, DCFH-DA Assay

Journal: Anatomy & Cell Biology

Article Title: Nicotinamide adenine dinucleotide phosphate oxidase inhibitor induces apoptosis on Epstein-Barr virus positive B lymphoma cells

doi: 10.5115/acb.20.277

Figure Lengend Snippet: Cell viability changes at 48 hours after Nox2 inhibitor, GSK2795039, treatment. Cells were seeded and treated with GSK2795039 at the indicated doses for 48 hours. Cell viability was analyzed using a WST-1 assay or trypan blue exclusion method. (A) Primary B cells, (B) Raji cells, (C) Ramos cells. Nox2, nicotinamide adenine dinucleotide phosphate oxidase 2; Primary B, primary B cells. The data shown are representative of three independent experiments (* P <0.05 vs. control, ** P <0.01 vs. control).

Article Snippet: Western blot was performed using the following primary antibodies: anti-human Nox2, anti-human Nox4 (BD Biosciences, San Diego, CA, USA), and anti-human β-actin antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: WST-1 Assay

Journal: Anatomy & Cell Biology

Article Title: Nicotinamide adenine dinucleotide phosphate oxidase inhibitor induces apoptosis on Epstein-Barr virus positive B lymphoma cells

doi: 10.5115/acb.20.277

Figure Lengend Snippet: PCR and western blot analyses of NADPH oxidase expression in primary B, Raji, and Ramos cells. (A) NADPH oxidase mRNA expression was verified by RT-PCR. β-actin was used as a loading control. Cells were harvested, and PCR amplification was performed as described as in the Materials and Methods. (B) Cells were harvested and cell lysates were extracted. The cell lysates were subjected to western blot using anti-human Nox2, -4, and β-actin antibodies. β-actin was used as a loading control. Results are representative of three independent experiments. NADPH, nicotinamide adenine dinucleotide phosphate; Nox2, NADPH oxidase 2; Nox4, NADPH oxidase 4; PCR, polymerase chain reaction; Primary B, primary B cells; RT, reverse-transcription.

Article Snippet: Western blot was performed using the following primary antibodies: anti-human Nox2, anti-human Nox4 (BD Biosciences, San Diego, CA, USA), and anti-human β-actin antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Western Blot, Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification, Polymerase Chain Reaction

Journal: Anatomy & Cell Biology

Article Title: Nicotinamide adenine dinucleotide phosphate oxidase inhibitor induces apoptosis on Epstein-Barr virus positive B lymphoma cells

doi: 10.5115/acb.20.277

Figure Lengend Snippet: Apoptosis analysis of Raji cells after Nox2 inhibitor, GSK2795039 treatment. Raji cells were treated with GSK2795039 for 48 hours. Cells were harvested, washed, and stained with FITC-conjugated Annexin V and PI as described in the Materials and Methods. Stained cells were analyzed by flow cytometry. The numbers in box represents the cell proportion in each quadrant. Data are representative of the three independent experiments. DMSO, control for GSK2795039; Negative, negative control; Nox2, nicotinamide adenine dinucleotide phosphate oxidase 2; PI, propidium iodide.

Article Snippet: Western blot was performed using the following primary antibodies: anti-human Nox2, anti-human Nox4 (BD Biosciences, San Diego, CA, USA), and anti-human β-actin antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Staining, Flow Cytometry, Negative Control

Journal: Anatomy & Cell Biology

Article Title: Nicotinamide adenine dinucleotide phosphate oxidase inhibitor induces apoptosis on Epstein-Barr virus positive B lymphoma cells

doi: 10.5115/acb.20.277

Figure Lengend Snippet: GSK2795039, the Nox2 inhibitor, mediated caspase-dependent apoptotic cell death in Raji cells in a dose-dependent manner. (A) Raji and Ramos cells were treated with GSK2795039 in various concentrations for 48 hours and then stained with Annexin V-FITC. Apoptotic cell populations were determined with flow cytometry. * and ** indicate P <0.05 and P <0.01, respectively, compared with the Ramos cell apoptotic population. (B) Western blot analysis of caspase activation in cells after GSK2795039 treatment. β-actin was used as a loading control. Cell graphs represent the mean±standard deviation of three independent experiments in triplicate. Red box represents meaningful changes of molecule expressions. Nox2, nicotinamide adenine dinucleotide phosphate oxidase 2.

Article Snippet: Western blot was performed using the following primary antibodies: anti-human Nox2, anti-human Nox4 (BD Biosciences, San Diego, CA, USA), and anti-human β-actin antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Staining, Flow Cytometry, Western Blot, Activation Assay, Standard Deviation

Journal: Anatomy & Cell Biology

Article Title: Nicotinamide adenine dinucleotide phosphate oxidase inhibitor induces apoptosis on Epstein-Barr virus positive B lymphoma cells

doi: 10.5115/acb.20.277

Figure Lengend Snippet: Effects of GSK2795039 on the translocation of Bax and release of Cyt c in Raji cells. After GSK2795039 treatment, cells were harvested and cell lysates were extracted. The cell lysates were separated into cytosolic and mitochondrial fractions, which were then analyzed by western blotting using anti-human Cyt c , bax, beta-tubulin, and Cox IV antibodies. Anti-Cox IV and anti-beta tubulin antibodies were used for fraction loading controls. Results are representative of three independent experiments. Cyt c, cytochrome c ; DMSO ctr, solvent control; GSK2795039, Nox2 inhibitor; NAC, N-acetyl-L-cysteine, reactive oxygen species scavenger; Negative, negative control without treatment; z-VAD, N-benzyloxycarbonyl-Val-Ala-Asp, a pan-caspase inhibitor.

Article Snippet: Western blot was performed using the following primary antibodies: anti-human Nox2, anti-human Nox4 (BD Biosciences, San Diego, CA, USA), and anti-human β-actin antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Translocation Assay, Western Blot, Negative Control

Journal: Anatomy & Cell Biology

Article Title: Nicotinamide adenine dinucleotide phosphate oxidase inhibitor induces apoptosis on Epstein-Barr virus positive B lymphoma cells

doi: 10.5115/acb.20.277

Figure Lengend Snippet: Inhibitory effects of z-VAD, NAC, and Bim inhibitor 1 on the changes of sub-G1 populations induced by GSK2795039 in Raji cells. To assess apoptotic cells, cells were stained with PI as described in the Materials and Methods. Cells were then analyzed by flow cytometry. The percentage represents the fraction of sub-G1 cells (M1) relative to the total number of cells. Results are representative of three independent experiments. DMSO ctr, solvent control; GSK2795039, Nox2 inhibitor; NAC, N-acetyl-L-cysteine, reactive oxygen species scavenger; Negative, negative control without treatment; z-VAD, N-benzyloxycarbonyl-Val-Ala-Asp, pan-caspase inhibitor.

Article Snippet: Western blot was performed using the following primary antibodies: anti-human Nox2, anti-human Nox4 (BD Biosciences, San Diego, CA, USA), and anti-human β-actin antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Staining, Flow Cytometry, Negative Control

Journal: Anatomy & Cell Biology

Article Title: Nicotinamide adenine dinucleotide phosphate oxidase inhibitor induces apoptosis on Epstein-Barr virus positive B lymphoma cells

doi: 10.5115/acb.20.277

Figure Lengend Snippet: Schematic diagram of the intracellular signaling mechanism during Nox2 inhibitor-induced apoptosis in Raji cells. The increase of Nox2 expression effectively causes ROS generation as an initial response in EBV-positive B lymphoma Raji cells. Both the Nox inhibitor and ROS scavenger NAC effectively inhibit the cellular response following ROS generation. Aberrant ROS-mediated Bim and Noxa activation and Mcl-1 down-regulation induce the translocation of Bax to the mitochondria membrane, resulting in mitochondrial membrane potential disruption. The Bax pores cause the release of Cyt c into the cytoplasm, resulting in caspase cascade activation and apoptosis. Cyt c, cytochrome c ; EBV, Epstein-Barr virus; NAC, N-acetyl-L-cysteine; Nox2, nicotinamide adenine dinucleotide phosphate oxidase 2; ROS, reactive oxygen species.

Article Snippet: Western blot was performed using the following primary antibodies: anti-human Nox2, anti-human Nox4 (BD Biosciences, San Diego, CA, USA), and anti-human β-actin antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA).

Techniques: Expressing, Activation Assay, Translocation Assay

Journal: International Journal of Molecular Sciences

Article Title: Uric Acid Induces Hepatocytes Ferroptosis Through HIF-2α/DMT1-Mediated Iron Overload

doi: 10.3390/ijms27062833

Figure Lengend Snippet: Hyperuricemia induces ferroptosis in hepatocytes. ( A – C ) Serum levels of uric acid (UA), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) in mice ( n = 6). ( D ) HE staining of mouse liver tissues ( n = 6). ( E – H ) Malondialdehyde (MDA) and lipid peroxidation (LPO) levels in mouse liver ( n = 6) and L02 hepatocytes ( n = 3). ( I ) DCFH-DA probe fluorescence in L02 cells ( n = 3). ( J – L ) Fe 2+ levels in mouse liver ( n = 6) and L02 cells ( n = 3). ( M , N ) Western blot analysis of NOX4 protein expression in mouse liver ( n = 6) and L02 cells ( n = 3). ( O ) CCK8 assay for L02 cell viability (0, 250, 500, 750, 1000 μM). ( P ) LDH activity in L02 cells treated with 750 μM uric acid for 48 h ( n = 3). ( Q ) Western blot analysis of GPX4 protein expression in L02 cells ( n = 3). ( R ) Transmission electron microscopy (TEM) images showing mitochondrial ultrastructure in mouse liver cells. ( S ) Mitochondrial membrane potential (MMP) in L02 cells detected by JC-1 probe ( n = 3). * p < 0.05, ** p < 0.01 vs. control group, ## p < 0.01, ### p < 0.001 vs. 250 µM group.

Article Snippet: Anti-HIF-2α antibody (Cat# ET7107-32)and anti-NOX4 antibody (Cat# EM1706-66) were obtained from HuaBio (Hangzhou, China).

Techniques: Staining, Fluorescence, Western Blot, Expressing, CCK-8 Assay, Activity Assay, Transmission Assay, Electron Microscopy, Membrane, Control

Journal: International Journal of Molecular Sciences

Article Title: Uric Acid Induces Hepatocytes Ferroptosis Through HIF-2α/DMT1-Mediated Iron Overload

doi: 10.3390/ijms27062833

Figure Lengend Snippet: Deferoxamine alleviates hyperuricemia-induced ferroptosis in hepatocytes. ( A ) Western blot analysis of NOX4 protein expression in L02 cells after deferoxamine treatment (n = 3). ( B ) NOX4 mRNA levels in L02 cells after deferoxamine treatment detected ( n = 3). ( C ) CCK-8 assay for L02 cell viability after deferoxamine treatment ( n = 3). ( D , E ) MDA and LPO levels in L02 hepatocytes after deferoxamine treatment ( n = 3). ( F , G ) Fe 2+ levels in L02 cells after deferoxamine treatment ( n = 3). ( H ) DCFH-DA probe fluorescence in L02 cells after deferoxamine treatment ( n = 3). ( I ) MMP in L02 cells after deferoxamine treatment detected by JC-1 probe ( n = 3). * p < 0.05, ** p < 0.01 vs. control group, # p < 0.05, ## p < 0.01 vs. model group.

Article Snippet: Anti-HIF-2α antibody (Cat# ET7107-32)and anti-NOX4 antibody (Cat# EM1706-66) were obtained from HuaBio (Hangzhou, China).

Techniques: Western Blot, Expressing, CCK-8 Assay, Fluorescence, Control

Journal: International Journal of Molecular Sciences

Article Title: Uric Acid Induces Hepatocytes Ferroptosis Through HIF-2α/DMT1-Mediated Iron Overload

doi: 10.3390/ijms27062833

Figure Lengend Snippet: HIF-2α inhibitor alleviates hyperuricemia-induced ferroptosis in hepatocytes. ( A , B ) Western blot analysis of HIF-2α and NOX4 protein expression in L02 cells after HIF-2α-IN-4 treatment ( n = 3). ( C ) NOX4 mRNA levels in L02 cells after HIF-2α-IN-4 treatment ( n = 3). ( D , E ) MDA and LPO levels in L02 hepatocytes after HIF-2α-IN-4 treatment ( n = 3). ( F ) DCFH-DA probe fluorescence in L02 cells after HIF-2α-IN-4 treatment ( n = 3). ( G ) Fe 2+ levels in L02 cells after HIF-2α-IN-4 treatment detected by FerroOrange probe ( n = 3). ( H ) MMP in L02 cells after HIF-2α-IN-4 treatment detected by JC-1 probe ( n = 3). * p < 0.05, ** p < 0.01 vs. control group, # p < 0.05 vs. model group.

Article Snippet: Anti-HIF-2α antibody (Cat# ET7107-32)and anti-NOX4 antibody (Cat# EM1706-66) were obtained from HuaBio (Hangzhou, China).

Techniques: Western Blot, Expressing, Fluorescence, Control

Journal: International Journal of Molecular Sciences

Article Title: Uric Acid Induces Hepatocytes Ferroptosis Through HIF-2α/DMT1-Mediated Iron Overload

doi: 10.3390/ijms27062833

Figure Lengend Snippet: HIF-2α knockdown alleviates hyperuricemia-induced ferroptosis in hepatocytes. ( A ) Western blot analysis of TFRC, DMT1, NOX4, and HIF-2α protein expression in L02 cells after HIF-2α knockdown ( n = 3). ( B – E ) HIF-2α, DMT1, TFRC, and NOX4 mRNA levels in L02 cells after HIF-2α knockdown detected by qRT-PCR ( n = 3). ( F ) Immunofluorescence staining of DMT1 and TFRC proteins in L02 cells after HIF-2α knockdown [Immunofluorescence staining showing TFRC (red), DMT1 (purple), and nuclei counterstained with DAPI (blue), n = 3]. ( G – I ) Fe 2+ , LPO and MDA levels in L02 cells after HIF-2α knockdown ( n = 3). ( J ) DCFH-DA probe fluorescence in L02 cells after HIF-2α knockdown ( n = 3). * p < 0.05, ** p < 0.01 vs. control group, # p < 0.05, ## p < 0.01 vs. model group, ns, not significant vs. model group.

Article Snippet: Anti-HIF-2α antibody (Cat# ET7107-32)and anti-NOX4 antibody (Cat# EM1706-66) were obtained from HuaBio (Hangzhou, China).

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