Journal: Mediators of inflammation

Article Title: Poldip2/Nox4 Mediates Lipopolysaccharide-Induced Oxidative Stress and Inflammation in Human Lung Epithelial Cells.

doi: 10.1155/2022/6666022

Figure Lengend Snippet: Figure 1: Poldip2 and Nox4 were upregulated in response to LPS in a dose/time-dependent manner in human lung epithelial cells. (a, b) A549 and Beas-2B cells were treated with the different doses of LPS (0–40 μg/mL for A549 cells, 0–10 μg/mL for Beas-2B cells) for 24 h, and the cell viability was analyzed by MTT assay. (c, d) Confluent cells were treated with different doses of LPS (0–20 μg/mL for A549 cells and 0–5 μg/mL for Beas-2B cells) for 12 h, and the protein levels of Poldip2 and Nox4 were detected by Western blotting and quantitatively analyzed. (e, f) Confluent cells were treated with LPS (10 μg/mL for A549 cells and 1 μg/mL for Beas-2B cells) for the different times (0–24 h), and the protein levels of Poldip2 and Nox4 were detected by Western blotting and quantitatively analyzed. Bars are mean ± standard deviation(SD) of 3 independent experiments. ∗∗∗P < 0:001 vs. 0 μg/mL or 0 h, ∗∗P < 0:01 vs. 0 μg/mL or 0 h, and ∗P < 0:05 vs. 0 μg/mL or 0 h.

Article Snippet: Primary antibodies of Nox4 were purchased from Novus (Novus, USA).

Techniques: MTT Assay, Western Blot, Standard Deviation

Journal: Mediators of inflammation

Article Title: Poldip2/Nox4 Mediates Lipopolysaccharide-Induced Oxidative Stress and Inflammation in Human Lung Epithelial Cells.

doi: 10.1155/2022/6666022

Figure Lengend Snippet: Figure 2: Poldip2 interacted with Nox4 induced by LPS with coimmunoprecipitation. (a, b) Confluent cells were treated with LPS (10 μg/ mL for A549 cells and 1 μg/mL for Beas-2B cells) for 12 h. Cell lysates were immunoprecipitated with normal rabbit IgG (isotype controls) or primary rabbit anti-Poldip2 antibody and immunoblotted with a mouse anti-Nox4 antibody or rabbit anti-Poldip2 antibody. Lysis buffer (Input) is a positive control in coimmunoprecipitation.

Article Snippet: Primary antibodies of Nox4 were purchased from Novus (Novus, USA).

Techniques: Immunoprecipitation, Lysis, Positive Control

Journal: Mediators of inflammation

Article Title: Poldip2/Nox4 Mediates Lipopolysaccharide-Induced Oxidative Stress and Inflammation in Human Lung Epithelial Cells.

doi: 10.1155/2022/6666022

Figure Lengend Snippet: Figure 3: Nox4 is required for Poldip2 to exert action in response to LPS. (a, b) Confluent A549 and Beas-2B cells were transfected with siRNA for 72 h, and the efficiency of Nox4 downregulation was verified by Western blotting and quantitatively analyzed. Following transfection, they were treated with LPS (10 μg/mL for A549 cells and 1 μg/mL for Beas-2B cells) for 12 h. (c, d) The protein levels of Poldip2, HO-1, and COX-2 were detected by Western blotting and quantitatively analyzed. (e–g, i) The expression of Poldip2 was detected by immunofluorescence analysis and laser confocal microscope. The cells were stained with Poldip2 (green) and counter stained with DAPI (blue) (magnification of 400x). (h, j) The NADPH enzymatic activity in cells was detected by spectrophotometer. (k, l) The production of intracellular ROS was detected by flow cytometry. (m–r) The expression levels of PGE2, TNF-α, and IL-1β in the supernatant were detected by ELISA. Bars are mean ± SD of 3 independent experiments. ns: not significant, ∗∗∗P < 0:001 vs. siCont, ∗∗P < 0:01 vs. siCont, ∗P < 0:05 vs. siCont, ###P < 0:001 vs. siCont + LPS, ##P < 0:01 vs. siCont + LPS, and #P < 0:05 vs. siCont + LPS.

Article Snippet: Primary antibodies of Nox4 were purchased from Novus (Novus, USA).

Techniques: Transfection, Western Blot, Expressing, Microscopy, Staining, Activity Assay, Spectrophotometry, Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Mediators of inflammation

Article Title: Poldip2/Nox4 Mediates Lipopolysaccharide-Induced Oxidative Stress and Inflammation in Human Lung Epithelial Cells.

doi: 10.1155/2022/6666022

Figure Lengend Snippet: Figure 4: Upregulation of Nox4 increased oxidative stress and inflammation. (a, b) Confluent A549 and Beas-2B cells were transfected with Nox4 plasmid or the empty vector for 72 h, and the efficiency of Nox4 upregulation was verified by Western blotting and quantitatively analyzed. Confluent shCont and shPoldip2 cells were cotransfected with Nox4 plasmid or the empty vector for 72 h. (c, d) The protein levels of Poldip2, HO-1, and COX-2 were detected by Western blotting and quantitatively analyzed. (e, f) The production of intracellular ROS was detected by flow cytometry. (g–l) The expression levels of PGE2, TNF-α, and IL-1β in the supernatant were detected by ELISA. Bars are mean ± SD of 3 independent experiments. ∗∗∗P < 0:001 vs. shCont + empty vector, ∗∗P < 0:01 vs. shCont + empty vector, ∗P < 0:05 vs. empty vector, ###P < 0:001 vs. shPoldip2 + empty vector, and ##P < 0:01 vs. shPoldip2 + empty vector.

Article Snippet: Primary antibodies of Nox4 were purchased from Novus (Novus, USA).

Techniques: Transfection, Plasmid Preparation, Western Blot, Cytometry, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Mediators of inflammation

Article Title: Poldip2/Nox4 Mediates Lipopolysaccharide-Induced Oxidative Stress and Inflammation in Human Lung Epithelial Cells.

doi: 10.1155/2022/6666022

Figure Lengend Snippet: Figure 6: Upregulation of Poldip2 increased LPS-induced oxidative stress and inflammation. (a, b) Confluent A549 and Beas-2B cells were transfected with the lentivirus upregulation vector for 72 h, and the efficiency of Poldip2 upregulation was verified by Western blotting and quantitatively analyzed. Following transfection, they were treated with LPS (10 μg/mL for A549 cells and 1 μg/mL for Beas-2B cells) for 12 h. (c, d) The protein levels of Nox4, HO-1, and COX-2 were detected by Western blotting and quantitatively analyzed. (e–g, i) The expression of Nox4 in the transfected cells was detected by immunofluorescence analysis and a laser confocal microscope. The cells were stained with Nox4 (green) and counter stained with DAPI (blue) (magnification of 400x). (h, j) The NADPH enzymatic activity in cells was detected by a spectrophotometer. (k, l) The production of intracellular ROS was detected by flow cytometry. (m–r) The expression levels of PGE2, TNF-α, and IL-1β in the supernatant were detected by ELISA. Bars are mean ± SD of 3 independent experiments. ∗∗∗P < 0:001 vs. LvCont, ∗∗P < 0:01 vs. LvCont, ∗P < 0:05 vs. LvCont, ###P < 0:001 vs. LvCont + LPS, ##P < 0:01 vs. LvCont + LPS, and #P < 0:05 vs. LvCont + LPS.

Article Snippet: Primary antibodies of Nox4 were purchased from Novus (Novus, USA).

Techniques: Transfection, Plasmid Preparation, Western Blot, Expressing, Microscopy, Staining, Activity Assay, Spectrophotometry, Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Mediators of inflammation

Article Title: Poldip2/Nox4 Mediates Lipopolysaccharide-Induced Oxidative Stress and Inflammation in Human Lung Epithelial Cells.

doi: 10.1155/2022/6666022

Figure Lengend Snippet: Figure 7: Effects of PI3K-AKT signaling on the activation of the Poldip2/Nox4-mediated pathway. Confluent A549 and Beas-2B cells were pretreated with 10 μmol/L LY294002 for 1 h followed by postincubation with LPS (10 μg/mL for A549 cells and 1 μg/mL for Beas-2B cells) for 12 h. (a, b) The protein levels of p-AKT, AKT, Poldip2, Nox4, HO-1, and COX-2 were detected by Western blotting and quantitatively analyzed. (c–h) The expression of Poldip2 and Nox4 in cells was detected by immunofluorescence analysis and a laser confocal microscope. The cells were stained with Poldip2 (green), Nox4 (green), and counter-stained with DAPI (blue) (magnification of 400x). Bars are mean ± SD of 3 independent experiments. ∗∗∗P < 0:001 vs. control, ∗∗P < 0:01 vs. control, ∗P < 0:05 vs. control, ###P < 0:001 vs. LPS, ##P < 0:01 vs. LPS, and #P < 0:05 vs. LPS.

Article Snippet: Primary antibodies of Nox4 were purchased from Novus (Novus, USA).

Techniques: Activation Assay, Western Blot, Expressing, Microscopy, Staining, Control

Journal: Journal of Cellular and Molecular Medicine

Article Title: Nox2 and Nox4 mediate tumour necrosis factor-α-induced ventricular remodelling in mice

doi: 10.1111/j.1582-4934.2011.01261.x

Figure Lengend Snippet: Gene symbol, assay ID and RefSeq for RT-PCR

Article Snippet: Mouse Nox2, Nox4 and ANP proteins were examined using purified anti-gp91phox (BD Biosciences, San Diego, CA, USA), anti-Nox4 antibody (Novus Biologicals, Littleton, CO, USA) and anti-ANP (Santa Cruz Biotechnology Inc.), following the procedures previously described [ ].

Techniques:

Journal: Journal of Cellular and Molecular Medicine

Article Title: Nox2 and Nox4 mediate tumour necrosis factor-α-induced ventricular remodelling in mice

doi: 10.1111/j.1582-4934.2011.01261.x

Figure Lengend Snippet: Upregulation of Nox2 and Nox4 mRNA and protein in ventricles of TNF-α-injected mice. Total RNA was extracted from the ventricular tissues of TNF-α-injected and control mice and Nox2 and Nox4 mRNA levels were examined using real-time PCR. Nox2 (A) and Nox4 mRNA levels (B) were shown. Nox2 and Nox4 protein levels were determined by immunoblotting. A representative of Nox2 and Nox4 proteins were shown (C). Densitometric analysis of Nox2 (D) and Nox4 protein (E) normalized with GAPDH were shown. C, control; T, TNF-α-injected mice. * P < 0.05.

Article Snippet: Mouse Nox2, Nox4 and ANP proteins were examined using purified anti-gp91phox (BD Biosciences, San Diego, CA, USA), anti-Nox4 antibody (Novus Biologicals, Littleton, CO, USA) and anti-ANP (Santa Cruz Biotechnology Inc.), following the procedures previously described [ ].

Techniques: Injection, Control, Real-time Polymerase Chain Reaction, Western Blot

Journal: Journal of Cellular and Molecular Medicine

Article Title: Nox2 and Nox4 mediate tumour necrosis factor-α-induced ventricular remodelling in mice

doi: 10.1111/j.1582-4934.2011.01261.x

Figure Lengend Snippet: Nox2 and Nox4 mediate TNF-α-induced upregulation of IL-1β and IL-6 genes and ROS production. Human adult cardiomyocytes were transfected with Nox2 and Nox4 siRNA using siPORT Amine transfection reagents. Total RNA was extracted from human adult cardiomyocytes 24 hrs after transfection. Human Nox2 and Nox4 mRNA levels were examined using real-time PCR. Nox2 (A) and Nox4 (B) mRNA levels after siRNA treatment were shown. In the other experiments, cells were treated with TNF-α (20 ng/ml) for 6 hrs at 37°C after treatment with Nox2 and Nox4 siRNA. IL-1β (C) and IL-6 mRNA (D) levels were examined with real-time PCR. TNF-α-induced ROS production in human adult cardiomyocytes was determined with CM-H 2 DCFDA following Nox2 and Nox4 siRNA treatment (E). Data were expressed as relative light unit (RLU) ± S.D. * P < 0.05 versus control values. # P < 0.05 versus TNF-α treatment values.

Article Snippet: Mouse Nox2, Nox4 and ANP proteins were examined using purified anti-gp91phox (BD Biosciences, San Diego, CA, USA), anti-Nox4 antibody (Novus Biologicals, Littleton, CO, USA) and anti-ANP (Santa Cruz Biotechnology Inc.), following the procedures previously described [ ].

Techniques: Transfection, Real-time Polymerase Chain Reaction, Control

Journal: PLoS ONE

Article Title: Long-Term Treatment with the Sodium Glucose Cotransporter 2 Inhibitor, Dapagliflozin, Ameliorates Glucose Homeostasis and Diabetic Nephropathy in db/db Mice

doi: 10.1371/journal.pone.0100777

Figure Lengend Snippet: (A, B) ROS production was detected by fluorescence microscopy using dihydroethidium (DHE) staining. ROS was predominantly localized in the interstitia of db/db mice, and was suppressed in the db/db +dapa groups compared with that in the db/db group. Original magnification, ×100. Data are mean ± SEM. * P <0.05. (C, D) Localization of Nox4 was detected by immunohistochemistry. The expression of Nox4 was predominantly localized in the interstitia of db/db mice, and was suppressed in the db/db +dapa groups compared with that in in the db/db group. Original magnification, ×100. Data are mean ± SEM. * P <0.05.

Article Snippet: Briefly, renal tissues were stained with Nox4 rabbit antibody (Novus Biologicals, Littleton, CO, USA) for 12 h at 4°C followed by HRP-conjugated goat anti-rabbit IgG antibody (Millipore).

Techniques: Fluorescence, Microscopy, Staining, Immunohistochemistry, Expressing

Journal: PLoS ONE

Article Title: Long-Term Treatment with the Sodium Glucose Cotransporter 2 Inhibitor, Dapagliflozin, Ameliorates Glucose Homeostasis and Diabetic Nephropathy in db/db Mice

doi: 10.1371/journal.pone.0100777

Figure Lengend Snippet: (A) ROS production was detected by fluorescence microscopy using dihydroethidium (DHE) staining. ROS production was not increased by mannitol (b) compared with normal glucose (a), but was increased by high glucose (c). High-glucose-induced ROS production was decreased by dapagliflozin pretreatment in a dose-dependent manner (d: 0.2 nM; e: 2.0 nM; f: 20.0 nM). (B) Densitometric quantification of ROS production. Data are mean ± SEM. * P <0.05 vs. high glucose; NG: normal glucose; Man: mannitol; HG: high glucose; dapa: dapagliflozin. Quantitative RT-PCR analysis of the expression of Nox4 (C), MCP-1 (D) and osteopontin (E) showed that dapagliflozin inhibited diabetes-induced inflammation in the kidney. mRNA levels were normalized against Atp5f1 expression. Data are mean ± SEM. * P <0.05.

Article Snippet: Briefly, renal tissues were stained with Nox4 rabbit antibody (Novus Biologicals, Littleton, CO, USA) for 12 h at 4°C followed by HRP-conjugated goat anti-rabbit IgG antibody (Millipore).

Techniques: Fluorescence, Microscopy, Staining, Quantitative RT-PCR, Expressing

Journal: International Journal of Molecular Sciences

Article Title: MicroRNA 148a Suppresses Tuberculous Fibrosis by Targeting NOX4 and POLDIP2

doi: 10.3390/ijms23062999

Figure Lengend Snippet: NOX4 and POLDIP2 mutually mediate HKMT-induced collagen-1A synthesis ( A ) NOX4, POLDIP2 and collagen-1A mRNA expression in the Met5A cells over time after HKMT treatment ( B ) NOX4, POLDIP2 and collagen-1A mRNA expression in the Met5A cells with HKMT treatment in the presence of SiCtrl and SiRNA targeting NOX4 (SiNOX4) ( C ) NOX4, POLDIP2 and collagen-1A mRNA expression in the Met5A cells with HKMT treatment in the presence of SiCtrl and SiRNA targeting POLDIP2 (SiPOLDIP2) ( D ) Interaction between NOX4 and POLDIP2 in Met5A cells after HKMT treatment confirmed by western blot and immunoprecipitation * significant difference ( p < 0.05) compared to control group (SiCtrl), ** significant difference ( p < 0.01) compared to (SiCtrl), # significant difference ( p < 0.05) compared to HKMT group (SiCtrl + HKMT), ## significant difference ( p < 0.01) compared to HKMT group (SiCtrl + HKMT).

Article Snippet: After deparaffinization, lung tissues were immunostained with NOX4 antibody (Cusabio, Wuhan, China).

Techniques: Expressing, Western Blot, Immunoprecipitation, Control

Journal: International Journal of Molecular Sciences

Article Title: MicroRNA 148a Suppresses Tuberculous Fibrosis by Targeting NOX4 and POLDIP2

doi: 10.3390/ijms23062999

Figure Lengend Snippet: miRNA expression in mesothelial cells with HKMT treatment. ( A ) miRNA expression in mPMCs of WT mouse and NOX4 KO mouse ( n = 3) ( B ) miRNA expression in mPMCs of WT mouse and NOX4 KO mouse following stimulation with and without HKMT. * significant difference ( p < 0.05) compared to control group (WT mPMC Non), # significant difference ( p < 0.05) compared to WT mPMC HKMT group ( C ) miRNA expression in human Met5A cells transfected with SiCtrl or SiNOX4 following stimulation with and without HKMT. ** significant difference ( p < 0.01) compared to (SiCtrl), # significant difference ( p < 0.05) compared to HKMT group (SiCtrl + HKMT), ## significant difference ( p < 0.01) compared to HKMT group (SiCtrl + HKMT).

Article Snippet: After deparaffinization, lung tissues were immunostained with NOX4 antibody (Cusabio, Wuhan, China).

Techniques: Expressing, Control, Transfection

Journal: International Journal of Molecular Sciences

Article Title: MicroRNA 148a Suppresses Tuberculous Fibrosis by Targeting NOX4 and POLDIP2

doi: 10.3390/ijms23062999

Figure Lengend Snippet: miR-148a decreased in the early stage of HKMT stimulation but increased as the exposure time increased. ( A ) NOX4, POLDIP2 mRNA expression in the Met5A cells pre-incubated with HKMT (10 ng/mL) for indicated times ( n = 3). ( B ) qRT-PCR analysis of miRNA expression in Met5A cells pre-incubated with HKMT (10 ng/mL) for indicated times ( n = 3). Results are presented as the mean ± SEM ( C ) NOX4, POLDIP2 mRNA expression in the WT-mPMCs pre-incubated with HKMT (10 ng/mL) for indicated times ( n = 3). ( D ) qRT-PCR analysis of miRNA expression in WT-mPMCs pre-incubated with HKMT (10 ng/mL) for indicated times ( n = 3). Results are presented as the mean ± SEM ( E ) The expression of circulating miRNA levels in tuberculous pleural effusion and transudate. * significant difference ( p < 0.05) compared to control group (Non), ** significant difference ( p < 0.01) compared to control group (Non).

Article Snippet: After deparaffinization, lung tissues were immunostained with NOX4 antibody (Cusabio, Wuhan, China).

Techniques: Expressing, Incubation, Quantitative RT-PCR, Control

Journal: International Journal of Molecular Sciences

Article Title: MicroRNA 148a Suppresses Tuberculous Fibrosis by Targeting NOX4 and POLDIP2

doi: 10.3390/ijms23062999

Figure Lengend Snippet: miR-148a can reduce tuberculous induced collagen-1A expression by regulating NOX4. ( A ) Levels of miR-148a in Met5A transfected with miR-148a mimics and miR-148 inhibitors in the presence or absence of HKMT treatment ( B ) NOX4 mRNA, POLDIP2 mRNA and collagen-1A mRNA in Met5A transfected with Ctrl mimics and miR-148a mimic in the presence or absence of HKMT treatment ( C ) NOX4 mRNA, POLDIP2 mRNA and collagen-1A mRNA in Met5A transfected with Ctrl inhibitor and miR-148 inhibitor in the presence or absence of HKMT treatment ( D ) NF-kB expression in Met5A transfected with miR-148a mimics in the presence or absence of HKMT treatment ( E ) ChiP assay of NF-kB binding in miR-148a promoter. Met5A cells were treated with or without HKMT for 3 h to collect the chromatin for immunoprecipitation with a specific anti-NOX4 antibody and a specific anti-POLDIP2 antibody. Quantitative data are expressed as mean ± SEM. * significant difference ( p < 0.05) compared to control group (Ctrl mimic or Ctrl inhibitor), ** significant difference ( p < 0.01) compared to control group (Ctrl mimic or Ctrl inhibitor), # significant difference ( p < 0.05) compared to HKMT group (Ctrl mimic or Ctrl inhibitor + HKMT), ## significant difference ( p < 0.01) compared to HKMT group (Ctrl mimic or Ctrl inhibitor + HKMT).

Article Snippet: After deparaffinization, lung tissues were immunostained with NOX4 antibody (Cusabio, Wuhan, China).

Techniques: Expressing, Transfection, Binding Assay, Immunoprecipitation, Control

Journal: International Journal of Molecular Sciences

Article Title: MicroRNA 148a Suppresses Tuberculous Fibrosis by Targeting NOX4 and POLDIP2

doi: 10.3390/ijms23062999

Figure Lengend Snippet: miR-148a prevents pulmonary fibrosis in mice ( A ) Schematic of experimental design ( B ) miR-148a expression ( C ) Immunoblots showing decreases of epithelial mesenchymal transition (EMT) markers, NOX4 and POLDIP2 in lung tissues of BCG treated mice with miR-148a mimics posttreatment ( D ) Total number of pleural cells in four groups ( E ) Photomicrographs from cytospin stained with Giemsa ( F ) Cytokine levels (IL-6, TNF-α and IFN-γ) assessed in the serum. * significant difference ( p < 0.05) compared to control group (PBS + Ctrl. mimic), # significant difference ( p < 0.05) compared to BCG + Ctrl. mimic group, ## significant difference ( p < 0.01) compared to BCG + Ctrl. mimic group.

Article Snippet: After deparaffinization, lung tissues were immunostained with NOX4 antibody (Cusabio, Wuhan, China).

Techniques: Expressing, Western Blot, Staining, Control

Journal: International Journal of Molecular Sciences

Article Title: MicroRNA 148a Suppresses Tuberculous Fibrosis by Targeting NOX4 and POLDIP2

doi: 10.3390/ijms23062999

Figure Lengend Snippet: miR-148a posttreatment regulates NOX4 expression and inhibits collagen-1A infiltrates in mouse lungs. ( A ) H&E staining, ×100 ( B ) Immunostaining of NOX4, ×100 ( C ) Masson Trichrome staining in mouse lung. ×100, ×200 ( D ) Immunofluorescence staining of NOX4 and mesothelin (markers for mesothelial cells). ×400 Light yellow areas represent colocalization of NOX4 (green) and mesothelin (red). DAPI,4′,6-Diamidino-2-phenylindole dihydrochloride.

Article Snippet: After deparaffinization, lung tissues were immunostained with NOX4 antibody (Cusabio, Wuhan, China).

Techniques: Expressing, Staining, Immunostaining, Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: MicroRNA 148a Suppresses Tuberculous Fibrosis by Targeting NOX4 and POLDIP2

doi: 10.3390/ijms23062999

Figure Lengend Snippet: Regulation of tuberculous fibrosis by miR-148a. Chronic HKMT exposure induces miR-148a expression. Initially, TB downregulates miRNA 148a, but finally upregulates miR-148a. Overexpression of miR-148a in mesothelial cells blocked NOX4 and POLDIP2 expression, thus preventing ECM deposition and lung fibrogenesis. NOX4 may also inhibit the expression of miR-148a, providing a negative feedback mechanism.

Article Snippet: After deparaffinization, lung tissues were immunostained with NOX4 antibody (Cusabio, Wuhan, China).

Techniques: Expressing, Over Expression

Journal: International Journal of Molecular Sciences

Article Title: MicroRNA 148a Suppresses Tuberculous Fibrosis by Targeting NOX4 and POLDIP2

doi: 10.3390/ijms23062999

Figure Lengend Snippet: Human and mouse sequences and accession numbers for primers (forward, FOR; reverse, REV) used in RT-PCR.

Article Snippet: After deparaffinization, lung tissues were immunostained with NOX4 antibody (Cusabio, Wuhan, China).

Techniques: