Journal: PeerJ

Article Title: Azoramide ameliorates cadmium-induced cytotoxicity by inhibiting endoplasmic reticulum stress and suppressing oxidative stress

doi: 10.7717/peerj.16844

Figure Lengend Snippet: (A and B) HK-2 and ARPE-19 cells were stained with DAPI, and representative images were captured under an inverted fluorescence microscope. The ratio of apoptotic nuclei to total nuclei was calculated and expressed as the apoptosis rate; each value is presented as the mean ± SD of at least three fields of view under the microscope. (C) HK-2 cells were pretreated with 20 μM azoramide for 5 h and coincubated with Cd (20 μM) for 24 h. PARP1 and C-PARP1 were detected by WB, and corresponding densitometric analysis was conducted ( n = 3). (D and E) HK-2 and ARPE-19 cells were preincubated with azoramide (20 μM) for 5 h and subsequently treated with or without Cd (20 μM) for 24 h. Cell apoptosis was assessed with flow cytometry after annexin V/PI staining ( n = 3 or 6). ** p < 0.01, *** p < 0.001 for the indicated comparisons.

Article Snippet: An antibody against light chain 3B (LC3B) (L7543; 1:1,000) was purchased from Sigma−Aldrich, and an antibody against poly ADP-ribose polymerase-1 (PARP1) (9532S; 1:1,000) was provided by Cell Signaling Technology (Danvers, MA, USA).

Techniques: Staining, Fluorescence, Microscopy, Flow Cytometry

Journal: PeerJ

Article Title: Azoramide ameliorates cadmium-induced cytotoxicity by inhibiting endoplasmic reticulum stress and suppressing oxidative stress

doi: 10.7717/peerj.16844

Figure Lengend Snippet: (A and B) HK-2 cells were preincubated with azoramide (20 μM) for 5 h and subsequently treated with or without Cd (20 μM) for 24 h. The expression levels of p-38 and p-JNK were detected by WB, and corresponding densitometric analysis was conducted (n = 3). (C and D) ARPE-19 cells were preincubated with azoramide (20 μM) for 5 h and subsequently treated with or without Cd (20 μM) for 24 h. Then, p-38 and p-JNK were detected by WB, and corresponding densitometric analysis was performed ( n = 3 or 4). (E and F) HK-2 cells were incubated with Cd (20 μM) in the presence or absence of the p38 MAPK inhibitor SB203580 (10 μM) or the JNK inhibitor SP600125 (10 μM) for 24 h. C-PARP1 was measured by WB, and corresponding densitometric analysis was conducted ( n = 3). (G–J) HK-2 and ARPE-19 cells were incubated with Cd (20 μM) in the presence or absence of the p38 MAPK inhibitor SB203580 (10 μM) or the JNK inhibitor SP600125 (10 μM) for 24 h, and then cell apoptosis rates were analyzed with flow cytometry ( n = 3). * p < 0.05, ** p < 0.01, *** p < 0.001 for the indicated comparisons.

Article Snippet: An antibody against light chain 3B (LC3B) (L7543; 1:1,000) was purchased from Sigma−Aldrich, and an antibody against poly ADP-ribose polymerase-1 (PARP1) (9532S; 1:1,000) was provided by Cell Signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Incubation, Flow Cytometry

Journal: Redox Biology

Article Title: Vitamin D receptor activation regulates microglia polarization and oxidative stress in spontaneously hypertensive rats and angiotensin II-exposed microglial cells: Role of renin-angiotensin system

doi: 10.1016/j.redox.2019.101295

Figure Lengend Snippet: The impacts of calcitriol on protein expression and activity of Nox in the hypertensive brain. Representative Western blot (A) and statistical graphs of protein expression of Nox2 (B), p-p47phox (C), p47phox (D) and Nox4 (E), and total Nox activity (F). 100 ng/kg calcitriol was administrated daily for 6 weeks in both control (Con) and SHR animals. Data are means ± SD (n = 7). ** p < 0.01 compared to control group. + p < 0.05, ++ p < 0.01 compared to SHR group.

Article Snippet: The following antibodies and concentrations were used over night at 4 °C; anti-VDR (Santa Cruz, sc-13133, 1:300), anti-AT1 (Proteintech, 25343-1-AP, 1:1000), anti-ACE (Abcam, ab254222, 1:1000), anti-ACE2 (Proteintech, 21115-1-AP, 1:1000), anti-MasR (Novus, NBP1-78444, 1:1000), anti-Nox2 (Santa Cruz, sc-130549, 1:800), anti-phospho-p47phox (Ser370) (Invitrogen, PA5-36863, 1:500), anti-p47phox (Santa Cruz, sc-17844, 1:200), Nox4 (Proteintech, 14347-1-AP, 1:1000).

Techniques: Expressing, Activity Assay, Western Blot, Control

Journal: Redox Biology

Article Title: Vitamin D receptor activation regulates microglia polarization and oxidative stress in spontaneously hypertensive rats and angiotensin II-exposed microglial cells: Role of renin-angiotensin system

doi: 10.1016/j.redox.2019.101295

Figure Lengend Snippet: The impacts of calcitriol treatment on Nox signaling and cellular superoxide generation in microglia BV2 cells. Representative western blots (A) and statistical graphs of protein expression of Nox2 (B), Nox4 (C), p-p47phox (D) and p47phox (E). NADPH oxidase activity (F) and ROS generation (DCF fluorescence intensity) (G). 1 μM calcitriol was pre-treated for 30min before exposure to 100 nM Ang II for 24 h. 1 μM A779 was added simultaneously with Ang II to block MasR. Data are means ± SD (n = 6). ** p < 0.01 compared to control group. ++ p < 0.01 compared to Ang II treated group. ## p < 0.05, ## p < 0.01 compared to Ang II + Calcitriol treated group.

Article Snippet: The following antibodies and concentrations were used over night at 4 °C; anti-VDR (Santa Cruz, sc-13133, 1:300), anti-AT1 (Proteintech, 25343-1-AP, 1:1000), anti-ACE (Abcam, ab254222, 1:1000), anti-ACE2 (Proteintech, 21115-1-AP, 1:1000), anti-MasR (Novus, NBP1-78444, 1:1000), anti-Nox2 (Santa Cruz, sc-130549, 1:800), anti-phospho-p47phox (Ser370) (Invitrogen, PA5-36863, 1:500), anti-p47phox (Santa Cruz, sc-17844, 1:200), Nox4 (Proteintech, 14347-1-AP, 1:1000).

Techniques: Western Blot, Expressing, Activity Assay, Fluorescence, Blocking Assay, Control

Journal: Frontiers in Oncology

Article Title: Hybrid peptide NTP-217 triggers ROS-mediated rapid necrosis in liver cancer cells by induction of mitochondrial leakage

doi: 10.3389/fonc.2022.1028600

Figure Lengend Snippet: Failure of NTP-217 to induce caspase-3 activation and apoptosis. (A) Hepa 1-6 cells were treated with different concentrations of NTP-217 for 4 h or with 12.5 μM NTP-217 for 24 h. Cells were stained with a caspase-3/7 assay kit and Hoechst 33258 (objective magnification, x4). (B) Quantification of apoptotic cells by counting the ratio of GreenNuc-positive and Hoechst 33258-positive cells. (C) Hepa 1-6 cells were incubated with NTP-217 alone or in the presence of 20 μM z-VAD-fmk for 4 h and then subjected to MTT assay. (D) PARP-1, cleaved PARP-1, caspase-3 and cleaved caspase-3 expression was measured using western blot analysis in Hepa 1-6 cells treated with NTP-217 for 4 or 24 h. (E) Quantification of PARP-1, cleaved PARP-1, caspase-3 and cleaved caspase-3 expression. Data are presented as the mean ± SD (n=3). * P<0.05, *** P<0.001, **** P<0.0001. ns, not significant; c, cleaved; Cas, caspase; PARP-1, poly (ADP-ribose) polymerase 1.

Article Snippet: The membranes were blocked with QuickBlock™ Blocking Buffer for Western Blot (Beyotime Institute of Biotechnology) and then incubated overnight at 4˚C with primary antibodies against Poly [ADP-ribose] polymerase 1 (1:5,000; cat. no. 66520-1; ProteinTech Group, Inc.), caspase-3 (1:10,000; cat. no. ab32499; Abcam), cleaved caspase-3 (1:1,000, cat. no. 9661, Cell Signaling Technology, Inc.) and GAPDH (1:10,000; cat. no. abs132004; Absin).

Techniques: Activation Assay, Staining, Incubation, MTT Assay, Expressing, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Free Radical-Mediated Protein Radical Formation in Differentiating Monocytes

doi: 10.3390/ijms22189963

Figure Lengend Snippet: ( A ) Hydroxyl radical imaging using CLSM. U-937 cells were incubated with 1 μM HPF and 250 nM PMA in the absence (upper panel) and presence (lower panel) of 100 µM apocynin. Images from DIC channel and HPF-ox fluorescence (green signal) are presented. ( B ) Western blot analysis of NADPH oxidase 4 expression in whole-cell homogenate samples treated with 150 nM and 250 nM of PMA for 72 h and their corresponding densitograms. ( C ) Western blot analysis of NADPH oxidase 4 expression in 250 nM PMA differentiated whole cell homogenates pre-treated with 50 µM and 100 µM apocynin and their corresponding densitograms.

Article Snippet: Recombinant anti-NADPH oxidase 4 antibody and rabbit polyclonal Anti-DMPO Nitrone adduct antibody were purchased from Abcam (Cambridge, CB2 0AX, UK).

Techniques: Imaging, Incubation, Fluorescence, Western Blot, Expressing

Journal: Cell Death and Differentiation

Article Title: Mitochondrial 4-HNE derived from MAO-A promotes mitoCa 2+ overload in chronic postischemic cardiac remodeling

doi: 10.1038/s41418-019-0470-y

Figure Lengend Snippet: a Model of AAV9-GFP or AAV9-ALDH2 (3 × 1011 vg/mouse) transduction in WT or MAO-A Tg mice. b Cardiac GSH content and c mitochondrial ALDH2 activity in WT and MAO-A Tg hearts at 12 weeks (n = 4). d Mitochondrial ALDH2 activity on cardiac homogenates of MAO-A Tg mice after AAV9 transduction (n = 7). e 4-HNE (Scale Bar = 100 μm) and Vinculin (Scale Bar = 50 μm) immunofluorescence, and interstitial fibrosis with Sirius Red (Scale Bar = 1 mm) on cardiac cryosections of mice after AAV9 transduction. f Quantification of 4-HNE fluorescence as % of total area (n = 6–7). g Quantification of cardiomyocytes area on at least 100 cells/mouse in three distinct regions of the left ventricle (n = 6–7). h Quantification of fibrosis as % of total area (n = 4–6). Echocardiographic measurements with (i) fractional shortening (%) and (j) systolic LV internal dimension on mice after AAV9 transduction (n = 6–7). Data are expressed as means ± sem (*p < 0.05, **p < 0.01, ***p < 0.001 vs WT GFP mice; #p < 0.05, ##p < 0.01 vs MAO-A Tg GFP mice).

Article Snippet: AAV9-cTnT-ALDH2 was made from the cDNA of mouse aldehyde dehydrogenase 2 (ALDH2) cloned into pCMV6-Entry vector (OriGene, Rockville, USA).

Techniques: Transduction, Activity Assay, Immunofluorescence, Fluorescence

Journal: Cell Death and Differentiation

Article Title: Mitochondrial 4-HNE derived from MAO-A promotes mitoCa 2+ overload in chronic postischemic cardiac remodeling

doi: 10.1038/s41418-019-0470-y

Figure Lengend Snippet: Echocardiographic parameters of WT and MAO-A Tg mice transduced with AAV9-cTnT-GFP or AAV9-cTnT-ALDH2 (M-MODE).

Article Snippet: AAV9-cTnT-ALDH2 was made from the cDNA of mouse aldehyde dehydrogenase 2 (ALDH2) cloned into pCMV6-Entry vector (OriGene, Rockville, USA).

Techniques: Transduction

Journal: Cell Death and Differentiation

Article Title: Mitochondrial 4-HNE derived from MAO-A promotes mitoCa 2+ overload in chronic postischemic cardiac remodeling

doi: 10.1038/s41418-019-0470-y

Figure Lengend Snippet: Immunoblots and quantifications of ALDH2, MAO-A, and 4-HNE protein adducts in (a) cardiac homogenates of SHAM or MI mice (n = 4) and (b) left ventricular myocardium of CTL (n = 4) or human ischemic cardiomyopathy patients (hICM) (n = 5). c Model of MI experiments with moclobemide treatment (20 mg/kg/day) or in mice with deletion of MAO-A in cardiomyocytes (MAO-A cKO). d Quantifications of 4-HNE protein adducts in mouse hearts after MI. (MAO cKO: n = 4 sham, n = 8 MI; Moclobemide: n = 5 sham, n = 5 MI). e Echocardiographic parameters of Ejection Fraction (EF, %). f Representative pictures of 3D-reconstructed hearts by LSFM with scar zone in blue in upper panel (Scale Bar = 2 mm) or Masson’s Trichrome staining in lower panel (Scale Bar = 1 mm). g Vinculin (Scale Bar = 50 μm) immunofluorescence staining with quantifications of mean cardiomyocyte area. (MAO-A cKO: n = 4 sham, n = 8 MI). h Immunoprecipitation experiments with MCU or 4-HNE in heart homogenates of mice subjected to 4 weeks ischemia (MI) in the presence of moclobemide (20 mg/kg/day) (n = 3 per group). i Representative nonreducing immunoblots showing MCU higher-order complex formation in heart homogenates of mice subjected to 4 weeks ischemia (MI) in the presence of moclobemide (20 mg/kg/day) (n = 3 per group). j Quantification of Ca2+ accumulation in isolated mitochondria of WT and MAO-A cKO mice subjected to 1 week ischemia (n = 5–7).

Article Snippet: AAV9-cTnT-ALDH2 was made from the cDNA of mouse aldehyde dehydrogenase 2 (ALDH2) cloned into pCMV6-Entry vector (OriGene, Rockville, USA).

Techniques: Western Blot, Staining, Immunofluorescence, Immunoprecipitation, Isolation

Journal: Journal of Cellular and Molecular Medicine

Article Title: Kallistatin attenuates endothelial senescence by modulating Let‐7g‐mediated miR‐34a‐ SIRT 1‐ eNOS pathway

doi: 10.1111/jcmm.13734

Figure Lengend Snippet: Kallistatin ( KS ) suppresses H 2 O 2 ‐induced oxidative stress and inflammation in human endothelial cells. Representative images of superoxide formation determined by fluorescence dye DCF (A), and quantitative analysis of DCF fluorescence (B). NADPH oxidase activity (C). mRNA levels of NADPH oxidase 4 (D), VCAM ‐1 (E), ICAM ‐1 (F), IL ‐6 (G) and miR‐34a (H) analysed by qRT ‐ PCR . Values are expressed as mean ± SEM. (n = 3 in each group). * P < .05 vs control, # P < .05 vs H 2 O 2 group

Article Snippet: The following primers were used: 18S (Hs99999901_s1), p16 INK4a (Hs00923894_m1), eNOS (Hs01574659_m1), SIRT1 (Hs01009006_m1), catalase (Hs00156308_m1), PAI‐1 (Hs01126606_m1), superoxide dismutase (SOD)‐2 (Hs00167309_m1), ICAM‐1 (Hs00277001_m1), interleukin (IL)‐6 (Hs00174131_m1), VCAM‐1 (Hs00365486_m1), NADPH oxidase 4 (Hs00418356_m1), miR‐34a (002316), Let‐7g (002282), mouse GAPDH (Mm99999915_g1), mouse eNOS (Mm00435217_m1), mouse SIRT1 (Mm00490758_m1), mouse catalase (Mm00437992_m1), mouse SOD‐1 (Mm01344233_g1), mouse VCAM1 (Mm01320970_m1), mouse IL‐6 (Mm00446190_m1), mouse ICAM‐1 (Mm00516024_g1), mouse miR‐34a (465771), mouse kallistatin (Mm00434669_m1), mouse PAI‐1 (Mm00435858_m1), mouse p16 INK4a (Mm00494449_m1).

Techniques: Fluorescence, Activity Assay, Quantitative RT-PCR, Control

Journal: Nature Communications

Article Title: Type I interferons affect the metabolic fitness of CD8 + T cells from patients with systemic lupus erythematosus

doi: 10.1038/s41467-021-22312-y

Figure Lengend Snippet: a Representative oxygen consumption rate (OCR) (left panels) and summary graphs (right panels) of sorted CD4 + and CD8 + T cells from controls, IFN-Neg and IFN-High SLE patients during a mitochondrial stress test. Spare respiratory capacity (SRC) was normalized to basal level of each individual. Oligomycin, carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), and rotenone/antimycin A (R/A) were added to the cells as indicated. Each symbol represents an individual (HC n = 12; IFN-Neg n = 7; IFN-High n = 8). b PBMCs from IFN-Neg and IFN-High SLE patients and HC were rested in culture for 48 h followed by staining with Annexin V and propidium iodide (PI). Representative flow cytometry plots of CD3+ CD8 + gated cells and summary graphs. Each symbol represents an individual (HC n = 11; IFN-Neg n = 11; IFN-High n = 12) a , b Data presented as mean ± S.E.M. One-way ANOVA, only significant differences are indicated, HC healthy controls. Source data for this figure are provided as a Source Data file.

Article Snippet: Afterwards, sort-purified CD8 + T cells were re-plated at 2.0 × 10 5 cells per well and re-stimulated with Gibco Dynabeads Human T-Activator CD3/CD28 (beads to cell ratio as indicated).

Techniques: Staining, Flow Cytometry

Journal: Nature Communications

Article Title: Type I interferons affect the metabolic fitness of CD8 + T cells from patients with systemic lupus erythematosus

doi: 10.1038/s41467-021-22312-y

Figure Lengend Snippet: a , b Purified CD8 + T cells from healthy donors treated with or without αCD3/CD28 beads in the presence or absence of 1000U/ml IFNα for 7 days. Mitochondria-encoded gene expression ( n = 5–7) ( a ) and changes in mitotracker stainings ( n = 6–7) ( b ) are shown. c , d PBMCs from healthy donors treated with or without αCD3/CD28 beads in the presence or absence of 1000U/ml IFNα for 7 days. CD8 + T cells were FACs-sorted and analysed using the extracellular flux assay. c Graphs showing the basal and maximal OCR levels under the different experimental conditions as indicated. Spare respiratory capacity (SRC) was normalized to basal level of each individual ( n = 7–8). d Oxidative response of FACs-sorted CD8 + T cells upon re-stimulation with anti-CD3/CD28 beads or PMA/I injected during the extracellular flux assay. A representative graph (left panel) and levels (right panels) at different time points normalised to basal level of each individual are shown ( n = 6). a – d Data presented as mean ± S.E.M. Each symbol represents one donor. a – c Two-tailed Wilcoxon matched-pairs signed rank test was utilized, d two-way ANOVA; only significant differences are indicated; MFI mean fluorescence intensity, MTG MitoTracker green, MTDR membrane potential dependent-Mitotracker Deep Read, MT-ND3 mitochondrially encoded NADH:Ubiquinone oxidoreductase core subunit 3, MT-CYTB mitochondrially encoded cytochrome B, PMA/I phorbol12-myristate13-acetate/Ionomycin. Source data for this figure are provided as a Source Data file.

Article Snippet: Afterwards, sort-purified CD8 + T cells were re-plated at 2.0 × 10 5 cells per well and re-stimulated with Gibco Dynabeads Human T-Activator CD3/CD28 (beads to cell ratio as indicated).

Techniques: Purification, Expressing, XF Assay, Injection, Two Tailed Test, Fluorescence

Journal: Nature Communications

Article Title: Type I interferons affect the metabolic fitness of CD8 + T cells from patients with systemic lupus erythematosus

doi: 10.1038/s41467-021-22312-y

Figure Lengend Snippet: PBMCs from HC were stimulated with αCD3/CD28 beads in the presence or absence of 1000U/ml of IFNα for 7 days. a αCD3/CD28 beads were removed from the culture and cells were left in culture for additional 48 h. Annexin V and propidium iodide (PI) staining of gated CD3 + CD8 + cells was used to detect spontaneous cell death. Data shown are mean ± S.E.M of fold-change of each individual sample exposed to αCD3/CD28 beads only. Each dot represents one donor ( n = 7). b , c αCD3/CD28 beads were removed from the culture and cells were rested overnight before labelling with CFSE and restimulation with αCD3/CD28 beads. b Representative flow plots of Annexin V staining and CFSE dilution (CD3 + CD8 + gated cells) at day 3 after re-stimulation (beads to cell ratio indicated). Percentages of Annexin V + CFSE low CD8 + T cells ( n = 5–7). c Percentages of CD8 + T cells positive for CD69, CD25, and CD107a at 4 h of post restimulation (beads to cell ratio = 1:2). Each symbol represents one donor ( n = 9). a – c Two-tailed Wilcoxon matched-pairs signed rank test; only significant differences are indicated; HC healthy controls. Source data for this figure are provided as a Source Data file.

Article Snippet: Afterwards, sort-purified CD8 + T cells were re-plated at 2.0 × 10 5 cells per well and re-stimulated with Gibco Dynabeads Human T-Activator CD3/CD28 (beads to cell ratio as indicated).

Techniques: Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Type I interferons affect the metabolic fitness of CD8 + T cells from patients with systemic lupus erythematosus

doi: 10.1038/s41467-021-22312-y

Figure Lengend Snippet: a Correlation between enrichment score for KEGG’s NAD metabolic pathway and Type I IFN signalling using GSVA package. Data obtained from the transcriptomic analysis of the SLE cohort. Pearson correlation coefficient ( r 2 ) and P -value are shown ( n = 34). b Gene expression of NAD-consuming enzymes (CD38; PARP9, PARP10, and PARP12) in CD8 + T cells from IFN-Neg (n = 6), IFN-High ( n = 15) SLE patients, and HC ( n = 11). Normalized read counts are shown. c , d Purified CD8 + T cells from HC were treated with IL-2 (10U/ml) and αCD3/CD28 with or without 1000U/ml IFNα for 7 days. c CD38 expression was measured using flow cytometry. MFI is shown ( n = 7). d NAD+/NADH ratio measured using NAD+/NADH Assay Kit (Abcam) ( n = 7). e – g Purified CD8 + T cells were stimulated as in c with or without addition of 1 mM NMN from day 3 to day 7. At day 7 CD8 + T cells were isolated and rested overnight. e NAD+/NADH ratio ( n = 7). f OCR levels 1 and 2 h after restimulation with αCD3/CD28 beads injected during the extracellular flux assay under the different experimental conditions as indicated. Data normalized to basal level of each individual ( n = 8). g Representative flow plots (left panels) of Annexin V and PI staining (CD3 + CD8 + gated cells) after αCD3/CD28 restimulation for 3 days are shown. Percentage of live cells (Annexin V − /PI − ) and apoptotic cells (Annexin V + ) under the different experimental conditions (right panels) are shown ( n = 9). a – g Data presented as mean ± S.E.M. Each symbol represents one donor. b Two-way ANOVA; c – f Two-tailed Wilcoxon matched-pairs signed rank test was utilized; g Matched-pairs One-Way ANOVA; Only significant data are indicated; MFI mean fluorescence intensity, PARP poly(ADP-ribose) polymerases, NAD nicotinamide adenine dinucleotide, NMN β-Nicotinamide mononucleotide, OCR oxygen consumption ratio, PI propidium iodide. Source data for this figure are provided as a Source Data file.

Article Snippet: Afterwards, sort-purified CD8 + T cells were re-plated at 2.0 × 10 5 cells per well and re-stimulated with Gibco Dynabeads Human T-Activator CD3/CD28 (beads to cell ratio as indicated).

Techniques: Expressing, Purification, Flow Cytometry, Nad NADH Assay, Isolation, Injection, XF Assay, Staining, Two Tailed Test, Fluorescence

Journal: Cells

Article Title: Micro-RNAs Shuttled by Extracellular Vesicles Secreted from Mesenchymal Stem Cells Dampen Astrocyte Pathological Activation and Support Neuroprotection in In-Vitro Models of ALS

doi: 10.3390/cells11233923

Figure Lengend Snippet: MSc-derived EVs positively modulate the Nrf2-NQO1 antioxidant pathway in mouse SOD1 G93A and patient-derived astrocytes. ( a – e ) Effect of MSC-derived EVs on oxidative stress in mouse SOD1 G93A astrocytes. ( a ) Representative WB images for Nrf2 and NQO1 in WT astrocytes, SOD1 G93A astrocytes and EV-treated SOD1 G93A astrocytes. ( b , c ) Quantitative analyses of WB experiments for Nrf2 and NQO1. Data are presented as means ± SEM of n = 3–4 (referred to Nrf2) and n = 3–5 (referred to NQO1) independent experiments, run in triplicate; statistical significance for p < 0.05 (* p < 0.05 vs. WT; Nrf2 F (2,8) = 5.307; NQO1 F (2,9) = 0.716; one-way ANOVA, followed by Bonferroni post hoc test). ( d ) Representative confocal microscopy images for GAPDH (red fluorescence), Nrf2 (green fluorescence) and DAPI (blue fluorescence), in WT, SOD1 G93A astrocytes and EV-treated SOD1 G93A astrocytes. Scale bar: 100 µm. ( e ) Quantitative representation of fluorescence intensity related to the nucleus/cytosol cellular localization ratio. Data are presented as means ± SEM of n = 3–5 independent experiments, run in triplicate; statistical significance for p < 0.05 (* p < 0.001 vs. WT; # p < 0.001 vs. untreated SOD1 G93A astrocytes; F (2,6) = 307.883; one-way ANOVA, followed by Bonferroni post hoc test). ( f – j ) Effect of MSC-derived EVs in human iAstrocytes. ( f ) Representative immunofluorescence images of Nrf2 (green fluorescence) and NQO1 (orange fluorescence) in untreated iAstrocytes and iAstrocytes exposed to human MSC-derived EVs. Scale bar: 100 µm. ( g ) Representative immunofluorescence images of reactive oxygen species stained with CellROX ® probe (orange fluorescence) in controls (CTR), untreated iAstrocytes and iAstrocytes exposed to human MSC-derived EVs. Scale bar: 100 µm. ( h ) Quantification of fluorescence for total Nrf2 expression in controls (CTR), C9orf72, and SOD1 patients, treated or not with human MSC-derived EVs. ( i ) Quantification of fluorescence for total NQO1 expression. Data are expressed as means ± SEM of n = 3 independent experiments. Statistical significance for p < 0.05 (* p < 0.05 vs. untreated iAstrocytes; F (2,17) = 42.335 for Nrf2; F (2,17) = 26.163 for NQO1; two-way ANOVA with Sidak’s multi comparison test). ( j ) Quantitative analyses of reactive oxygen species stained with CellROX ® probe, in controls (CTR), C9orf72, and SOD1 patients treated or not with EVs. Data are expressed as means ± SEM, n = 3 independent experiments, including two technical replicates per experiment. Statistical significance for p < 0.05 (* p < 0.05 vs. untreated iAstrocytes; F (2,17) = 6.605; two-way ANOVA with Sidak’s multi comparison test).

Article Snippet: Cells were fixed at day 8 with 4% paraformaldehyde for 10 min, followed by PBS washing and blocking with 5% donkey serum in 0.05% Triton-X/PBS for 30 min. Anti-nuclear factor erythroid 2–related factor 2 (Nrf2) (rabbit polyclonal, Abcam ab31163) and goat anti-NAD(P)H Quinone Dehydrogenase 1 (NQO1) (Abcam ab2346) antibodies were added at a dilution of 1:200 in 1% donkey serum/0.05% Triton-X/PBS and the cells were incubated overnight at 4 °C, washed with PBS, and incubated with goat anti-rabbit-488 (Abcam, Cambridge, UK, ab150077) and donkey anti-goat-555 (Abcam, Cambridge, UK, ab175704) antibodies, diluted at 1:500 at room temperature for 60 min.

Techniques: Derivative Assay, Confocal Microscopy, Fluorescence, Immunofluorescence, Staining, Expressing, Comparison

Journal: Cells

Article Title: Micro-RNAs Shuttled by Extracellular Vesicles Secreted from Mesenchymal Stem Cells Dampen Astrocyte Pathological Activation and Support Neuroprotection in In-Vitro Models of ALS

doi: 10.3390/cells11233923

Figure Lengend Snippet: EV-shuttled miRNAs modulate the pathological reactive phenotype, Nrf2 expression, and neurotoxicity in SOD1 G93A astrocytes and iAstrocytes. Graphical representation of ( a ) MAP3K8/MK2, ( b ) MAPK11 and ( f ) Nrf2 pathways. RT-qPCR of ( c ) Map3k8, ( d ) Mapkapk2, and ( e ) Mapk11 expression in WT, SOD1 G93A astrocytes + Hiperfect, SOD1 G93A astrocytes + scrambled miRNAs, and SOD1 G93A astrocytes + miRNAs. Data are presented as means ± SEM of n = 3 independent experiments, run in triplicate; statistical significance for p < 0.05 (* p < 0.05, vs. WT; # p < 0.01 vs. SOD1 G93A astrocytes + Hiperfect; § p < 0.01 vs. SOD1 G93A astrocytes + scrambled miRNA; F (6,14) = 11.738, F (6,14) = 6.321 and F (6,14) = 14.786 for Map3k8, Mapkapk2 and Mapk11, respectively; one-way ANOVA followed by Bonferroni post hoc test). ( g ) Representative images for GAPDH (red fluorescence), Nrf2 (green fluorescence) and DAPI (blue fluorescence), in WT, SOD1 G93A astrocytes, and SOD1 G93A astrocytes treated with miRNAs. Scale bar: 100 µm. ( h , i ) Quantitative representation of fluorescence intensity (see ) for ( h ) Nrf2 total expression and ( i ) Nrf2 cellular localization expressed as nuclear/cytoplasm ratio. Data are presented as means ± SEM of n = 3 independent experiments (referred to Nrf2 total expression) and n = 3–6 independent experiments (referred to Nrf2 cellular localization); statistical significance for p < 0.05 (* p < 0.001 vs. WT; # p < 0.001 vs. SOD1 G93A astrocytes + HighPerfect; § p < 0.001 vs. SOD1 G93A astrocytes + scrambled miRNA; Nrf2/GAPDH, F (4,10) = 7.507; Nrf2 nucleus/cytoplasm, F (4,10) = 214,640; two-way ANOVA followed by Bonferroni post hoc test). ( j ) RT-qPCR validation of Nrf2 and NQO1 expression in control iAstrocytes after miR-29b3P transfection. Data are presented as means ± SEM of n = 3 independent experiments; statistical significance for * p < 0.05 vs. control iAstrocytes (NQO1, t = −4.882; Nrf2, t = −0.0197; two-tailed Student’s t -test). ( k ) Quantification of MN survival expressed as the percentage of MNs with axon (calculated respect to the total number of MNs at day 1) after 3 days of co-culture with untreated iAstrocytes or iAstrocytes transfected with miRNA mimic miR-29b3P. Data are presented as means ± SEM of n = 3 biological replicates; statistical significance * p < 0.05 vs. untreated iAstrocytes; two-tailed Student’s t -test.

Article Snippet: Cells were fixed at day 8 with 4% paraformaldehyde for 10 min, followed by PBS washing and blocking with 5% donkey serum in 0.05% Triton-X/PBS for 30 min. Anti-nuclear factor erythroid 2–related factor 2 (Nrf2) (rabbit polyclonal, Abcam ab31163) and goat anti-NAD(P)H Quinone Dehydrogenase 1 (NQO1) (Abcam ab2346) antibodies were added at a dilution of 1:200 in 1% donkey serum/0.05% Triton-X/PBS and the cells were incubated overnight at 4 °C, washed with PBS, and incubated with goat anti-rabbit-488 (Abcam, Cambridge, UK, ab150077) and donkey anti-goat-555 (Abcam, Cambridge, UK, ab175704) antibodies, diluted at 1:500 at room temperature for 60 min.

Techniques: Expressing, Quantitative RT-PCR, Fluorescence, Biomarker Discovery, Control, Transfection, Two Tailed Test, Co-Culture Assay

Journal: Communications Biology

Article Title: βA1-crystallin regulates glucose metabolism and mitochondrial function in mouse retinal astrocytes by modulating PTP1B activity

doi: 10.1038/s42003-021-01763-5

Figure Lengend Snippet: a , b Plots from seahorse analysis using the mitostress assay showing time-dependent changes in metabolic flux upon treatment with mitochondrial respiration blockers oligomycin, Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), Rotenone/Antimycin A at particular time points (indicated by arrows). Y -axis denotes oxygen consumption rate (OCR; pmole/min/μg) and X -axis represents time (minutes) for a untreated (cultured in 5 mM d -Glucose) WT, βA3 KO, and βA1 KD mouse astrocytes or b astrocytes exposed to either mannitol (30 mM for 6 h) or high glucose (HG; 30 mM for 6 h) respectively, or βA1 astrocytes transfected with mCherry-βA1 overexpression construct for 48 h or treated with 10 μM of PTP1B inhibitor (MSI-1436) for 1 h prior to HG (30 mM) exposure for 6 h; n = 4. Reduced mitochondrial function is shown by decreased c ATP-linked respiration and d maximal respiration, in untreated or mannitol (30 mM for 6 h) and HG (30 mM for 6 h)-exposed βA1 KD astrocytes, compared to WT cells. WT and βA3 KO astrocytes treated with HG showed an increase in both c ATP-linked respiration and d maximal respiration, compared to untreated cells. In βA1 KD astrocytes transfected with βA1-mCherry overexpression construct for 48 h or treated with 10 μM of PTP1B inhibitor (MSI-1436) for 1 h prior to HG (30 mM) exposure for 6 h, the levels of c ATP-linked respiration and d maximal respiration were partially rescued; n = 4. * P < 0.05, ** P < 0.01. e , f Flow cytometry histograms and graph for MitoSox fluorescence (Alexa fluor (AF)-555), g Nox1 gene expression, h Nox2 gene expression and i superoxide release in βA1 KD astrocytes either untreated or exposed to mannitol (30 mM for 6 h) showed increased levels of e , f mROS, g Nox1 along with h Nox2 gene expression and i superoxide release, that increased further with HG (30 mM for 6 h) exposure, relative to WT cells ( e – h ). βA1-crystallin overexpression (using βA1-mCherry construct) or PTP1B inhibition in βA1 KD astrocytes followed by treatment with HG, reduced the elevated levels of e , f mROS, g Nox1 , and h Nox2 gene expression and i superoxide release in βA1 KD cells. n = 4. * P < 0.05, ** P < 0.01.

Article Snippet: qRT-PCR was performed as previously described using Taqman probes (Life Technologies, USA) for mouse Nox1 (Cat# Mm00549170_m1), Nox2 (Cat# Mm00516005_m1), Lepr (Cat# Mm00440181_m1), and Jak2 (Cat# Mm01208489_m1).

Techniques: Cell Culture, Transfection, Over Expression, Construct, Flow Cytometry, Fluorescence, Gene Expression, Inhibition

Journal: PLoS Pathogens

Article Title: A Cytoplasmic Tail Determinant in HIV-1 Vpu Mediates Targeting of Tetherin for Endosomal Degradation and Counteracts Interferon-Induced Restriction

doi: 10.1371/journal.ppat.1002609

Figure Lengend Snippet: ( A ) HT1080 cells stably expressing tetherin-HA were infected with VSV-G-pseudotyped HIV-1 wt, HIV-1 delVpu or HIV-1 Vpu ELV at an MOI of 2. 48 h post infection, cells were harvested and subjected to SDS-PAGE and analyzed by Western blotting for tetherin-HA, Vpu and Hsp90, and analyzed by LiCor quantitative imager. Relative tetherin-HA levels are indicated below each lane. The blot shown is a representative example of 3 independent experiments. ( B ) 293T cells were transfected with pCR3.1 Vpu-HA, Vpu 2/6A-HA, or Vpu ELV-HA in combination with pCR3.1 myc-β-TrCP2. 48 h post transfection, cells were lysed and immunoprecipitated with anti-HA antibody. Lysates and precipitates were subjected to SDS-PAGE and analyzed by Western blotting for Vpu and myc-β-TrCP2, and analyzed by ImageQuant. The star represents an unspecific band. ( C ) Similarly, 293T cells were transfected with pCR3.1 HA-HRS in combination with Vpu-GFP or Vpu ELV-GFP expression constructs. Cell lysates were precipitated with an anti-HA antibody and analyzed as in C . ( D ) HeLa cells were co-transfected with pCR3.1 Vpu-HA or indicated Vpu mutant in combination with a GFP expression construct. Cell surface staining for endogenous tetherin was analyzed by flow cytometry 48 h post transfection. GFP positive cells were gated and tetherin levels (solid lines) were compared to those of untransfected HeLa cells (dotted lines). Numbers indicate median fluorescence intensities of surface tetherin on transfected cells. The solid peak in the upper middle histogram represents the binding of the isotype control. ( E ) Jurkat cells were infected with VSV-G-pseudotyped HIV-1 wt, HIV-1 delVpu, HIV-1 Vpu ELV or HIV-1 Vpu 2/6A at an MOI of 1. 48 h post infection, cells were stained for cell surface tetherin and intracellular p24CA, and analyzed by flow cytometry. Productively infected cells were identified by comparing with culture infected with the same MOI in the presence of 50 µM AZT to control for p24CA uptake of the inoculum .

Article Snippet: Immunoprecipitation was performed as described above and Western blot assays were performed using a polyclonal rabbit anti-HA antibody (Rockland) and an anti-Vpu antibody.

Techniques: Stable Transfection, Expressing, Infection, SDS Page, Western Blot, Transfection, Immunoprecipitation, Construct, Mutagenesis, Staining, Flow Cytometry, Fluorescence, Binding Assay, Control

Journal: Oncotarget

Article Title: Interleukin-4 and interleukin-13 increase NADPH oxidase 1-related proliferation of human colon cancer cells

doi: 10.18632/oncotarget.17494

Figure Lengend Snippet: A . IL-4 induced a dose-dependent increase in NOX1 expression; HT-29 cells were treated with IL-4 at different concentrations (0, 0.1, 1, 10, 50, 200 ng/ml) for 24 h. NOX1 levels were analyzed by quantitative RT-PCR with β-actin serving as the internal control. B . HT-29 cells were treated with IL-4 (50 ng/ml) for different lengths of time (0, 3, 6, 9, 12, 24, and 48 h); levels of NOX1 expression were determined using quantitative RT-PCR and Western analysis with β-actin serving as the internal control. C . IL-4 and IL-13 specifically induced the expression of NOX1 but not of other NOX family members. HT-29 cells were treated with IL-4 (left) or IL-13 (right) at a concentration of 50 ng/ml for 24 h. Quantitative RT-PCR was performed to evaluate the expression levels of the individual NOX family members and their accessory genes; expression levels were normalized to β-actin. D . NOX1 protein induced by exposure to IL-4 or IL-13 is localized to the plasma membrane. HT-29 cells were treated with IL-4 (left) or IL-13 (right) at a concentration of 50 ng/ml for 24 h. Western analysis was performed on subcellular fractions (cytosolic, membrane, nuclear and cytoskeletal) to determine the localization of NOX1. GAPDH, ATPase, and Lamin A/C were used as cytosolic, membrane, and nuclear markers, respectively. Data represented as mean ± SD values; ** = P < 0.01; *** = P < 0.001.

Article Snippet: Human primers : NOX1 (catalog no. Hs00246589_m1), β-actin (catalog no. Hs99999903_m1), NOXA1 (catalog no. Hs00611456_g1), NOXO1 (catalog no. Hs00376039_g1), NOX2-5, DUOX1 and DUOX2 (catalog nos.

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot, Concentration Assay, Clinical Proteomics, Membrane

Journal: Oncotarget

Article Title: Interleukin-4 and interleukin-13 increase NADPH oxidase 1-related proliferation of human colon cancer cells

doi: 10.18632/oncotarget.17494

Figure Lengend Snippet: A . The effect of IL-4 on the protein expression of NOX1 is demonstrated by Western analysis under both serum-free conditions and in the presence of 10% FBS for both parental HT-29 cells and a clonal line selected for expression of a scrambled shRNA (SC cells). Modest to no effect of IL-4 on NOX1 expression is shown for the Si6/G6 intermediate NOX1 knockdown clones, or for 6A cells (> 90% NOX1 knockdown), respectively. B . NOX1 protein levels correlate with relative superoxide production following IL-4 exposure. Compared to superoxide production in HT-29 and SC cells exposed to IL-4, superoxide levels were only modestly increased in Si6/G6 cells, and were not changed at all by IL-4 treatment in 6A cells. C . IL-4 treatment increases ROS production in HT-29 cells. HT-29 cells were treated with IL-4 for the indicated times, harvested, and stained with CM-H 2 -DCFDA. Flow cytometry was performed to evaluate intracellular ROS levels. Compared with parental HT-29 cells, exposure to IL-4 for 96 h shifted intracellular fluorescence intensity to the right, indicating higher ROS levels; treatment with IL-4 for 5 min, however, produced no shift in DCF fluorescence. Pretreatment with the flavin dehydrogenase and NOX inhibitor DPI produced a left shift in fluorescence intensity. D . IL-4 (50 ng/ml) promotes HT-29 and SC cell proliferation. Cell numbers were counted daily in the presence or absence of IL-4 for 4 days. After the 4-day IL-4 treatment, compared with corresponding controls, HT-29 and SA cells showed a nearly 2-fold increase in cell number; the 6A ( NOX1 knockdown) cells demonstrated lower levels of proliferation in the presence or absence of IL-4; the G6 cells (partial NOX1 knockdown) displayed modest growth and modest enhancement of proliferation in the presence of IL-4. E . IL-4R expression is independent of NOX1 expression. Irrespective of NOX1 status, there was no significant change in IL-4Rα expression as measured by Western analysis (left panel) or quantitative RT-PCR (right panel). GAPDH and β-actin served as the internal controls, respectively. Data represent the mean ± SD of at least 3 experiments. ** = P < 0.01; *** = P < 0.001.

Article Snippet: Human primers : NOX1 (catalog no. Hs00246589_m1), β-actin (catalog no. Hs99999903_m1), NOXA1 (catalog no. Hs00611456_g1), NOXO1 (catalog no. Hs00376039_g1), NOX2-5, DUOX1 and DUOX2 (catalog nos.

Techniques: Expressing, Western Blot, shRNA, Knockdown, Clone Assay, Staining, Flow Cytometry, Fluorescence, Produced, Quantitative RT-PCR

Journal: Oncotarget

Article Title: Interleukin-4 and interleukin-13 increase NADPH oxidase 1-related proliferation of human colon cancer cells

doi: 10.18632/oncotarget.17494

Figure Lengend Snippet: A . IL-4 exposure for 24 h increases expression of full-length NOX1 in HT-29 cells. PCR and subsequent DNA gel analysis were performed to detect NOX1-L and NOX1-S in IL-4-treated HT-29 cells. A NOX1-L construct was transiently transfected into HT-29 cells and used as the control 48 h after transfection. B . IL-4 treatment (50 ng/ml for 24 h) supports PMA-stimulated superoxide production in HT-29 cells. HT-29 cells 48 h after transient transfection with a NOX1-L construct served as a positive control. C . Cell cycle analysis was performed using analytical cytometry following BrdU labeling of HT-29 cells that had been treated for 24 h with 50 ng/ml of IL-4. HT-29 cells evaluated 48 h after transient transfection with NOX1-L were used as the control. The percentage of cells in the different phases of the cell cycle is shown in the table. D . IL-4 exposure (50 ng/ml for 24 h) increases the expression of NOX1 in WiDr cells. PCR and subsequent DNA gel analysis were performed to detect NOX1-L and NOX1-S in WiDr cells treated with IL-4. Transient transfection of NOX1-L into WiDr cells served as the control. E . NOX1-L rather than NOX1-S supports PMA-stimulated superoxide production in WiDr cells. WiDr cells treated with IL-4 (50 ng/ml for 24 h) or 48 h after transient transfection with a NOX1-L construct were evaluated for the extent of PMA-induced superoxide production. F . Cell cycle analysis was performed using analytical cytometry following BrdU labeling of WiDr cells that had been treated for 24 h with 50 ng/ml of IL-4. WiDr cells evaluated 48 h after transient transfection with NOX1-L were used as the control. The percentage of cells in the different phases of the cell cycle is shown in the table. G . Western analysis was performed on HT-29 and WiDr cells treated with IL-4 (50 ng/ml) for variable periods of time (0, 24, 96 h). Activated STAT6 and NOX1 levels were examined as indicators of IL-4 efficacy in these experiments; GAPDH served as the loading control. All data represent the mean ± SD of at least three experiments. *** = P < 0.001.

Article Snippet: Human primers : NOX1 (catalog no. Hs00246589_m1), β-actin (catalog no. Hs99999903_m1), NOXA1 (catalog no. Hs00611456_g1), NOXO1 (catalog no. Hs00376039_g1), NOX2-5, DUOX1 and DUOX2 (catalog nos.

Techniques: Expressing, Construct, Transfection, Control, Positive Control, Cell Cycle Assay, Cytometry, Labeling, Western Blot

Journal: Oncotarget

Article Title: Interleukin-4 and interleukin-13 increase NADPH oxidase 1-related proliferation of human colon cancer cells

doi: 10.18632/oncotarget.17494

Figure Lengend Snippet: A .- B . Knockdown of IL-4Rα blocks NOX1 expression at the mRNA A . and protein B . levels. HT-29 cells were transiently transfected with either of the two different IL-4Rα specific siRNAs and treated with IL-4 (50 ng/ml) 24 h later. Cells were harvested 24 h post-treatment and examined by quantitative RT-PCR A . or by Western analysis B .. β-actin and GAPDH served as the internal controls for RT-PCR or Western analysis, respectively. C . IL-4-induced NOX1 expression requires the presence of IL-4R. IL-4-induced NOX1 expression was abolished in HT-29 by the concurrent presence of an IL-4Rα neutralizing antibody in the absence of FBS, as shown by quantitative RT-PCR. D . IL-4 stimulates cell proliferation through the involvement of IL-4R. IL-4Rα antibody (1 ng/ml) significantly inhibited IL-4-enhanced HT-29 proliferation. Data represent the mean ± SD of three experiments. ** = P < 0.01; *** = P < 0.001.

Article Snippet: Human primers : NOX1 (catalog no. Hs00246589_m1), β-actin (catalog no. Hs99999903_m1), NOXA1 (catalog no. Hs00611456_g1), NOXO1 (catalog no. Hs00376039_g1), NOX2-5, DUOX1 and DUOX2 (catalog nos.

Techniques: Knockdown, Expressing, Transfection, Quantitative RT-PCR, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Oncotarget

Article Title: Interleukin-4 and interleukin-13 increase NADPH oxidase 1-related proliferation of human colon cancer cells

doi: 10.18632/oncotarget.17494

Figure Lengend Snippet: A .- B . JAK1 knockdown blocks IL-4-mediated enhancement of NOX1 expression at the mRNA A . and protein levels B .. HT-29 cells were transiently transfected with either of the two different JAK1-specific siRNAs and treated with IL-4 (50 ng/ml) 24 h later. Cells were harvested at 24 h post-treatment and examined by quantitative RT-PCR A . and Western analysis B .. β-actin served as the internal control. C . IL-4 treatment results in a transient increase in phosphorylated STAT1 and STAT5, and a sustained increase in phosphorylated STAT6. Western analysis was performed on IL-4 (50 ng/ml)-treated HT-29 cells harvested at different time points following initiation of IL-4 exposure. β-actin served as the loading control. ( D .- E .) STAT6 knockdown blocks IL-4-related stimulation of NOX1 expression at the mRNA D . and protein levels E .. HT-29 cells were transiently transfected with either of two different STAT6-specific siRNAs and treated with IL-4 24 h later. Cells were harvested at 24 h post-treatment and examined by quantitative RT-PCR D . and Western analysis E .. F . PTP activity was evaluated in HT-29 and WiDr cells following exposure to IL-4 (50 ng/ml) for the indicated times. PTP levels were determined by measuring the dephosphorylation of a standard phosphopeptide using malachite green as the detector. β-actin served as the internal control. Data represent the mean ± SD of three experiments. *** = P < 0.001.

Article Snippet: Human primers : NOX1 (catalog no. Hs00246589_m1), β-actin (catalog no. Hs99999903_m1), NOXA1 (catalog no. Hs00611456_g1), NOXO1 (catalog no. Hs00376039_g1), NOX2-5, DUOX1 and DUOX2 (catalog nos.

Techniques: Knockdown, Expressing, Transfection, Quantitative RT-PCR, Western Blot, Control, Activity Assay, De-Phosphorylation Assay, Phospho-proteomics

Journal: Oncotarget

Article Title: Interleukin-4 and interleukin-13 increase NADPH oxidase 1-related proliferation of human colon cancer cells

doi: 10.18632/oncotarget.17494

Figure Lengend Snippet: A . IL-4 triggers GATA3 nuclear translocation demonstrated by Western analysis of HT-29 cells treated for 24 h with IL-4. GAPDH and Lamin A/C were used as the cytosolic and the nuclear protein loading controls, respectively. B .- C . GATA3 knockdown blocks increased NOX1 mRNA expression following IL-4 stimulation (50 ng/ml) at the mRNA B . and protein levels C .. HT-29 cells were transiently transfected with either of two different GATA3-specific siRNAs and treated with IL-4 24 h later. Cells were harvested 24 h following IL-4 treatment and examined by quantitative RT-PCR B . and Western analysis C .. β-actin and GAPDH served as the internal controls. D . The serine residues of GATA3 are phosphorylated following IL-4 stimulation. Immunoprecipitation of GATA3 and subsequent Western analysis to determine the extent of serine phosphorylation were performed on HT-29 cells treated with solvent or IL-4 (50 ng/ml for 24 h). E . Serine-phosphorylation of GATA3 is a prerequisite for IL-4-induced NOX1 expression. Western analysis was performed on HT-29 cells expressing either intact GATA3 or GATA3 S308A . F . Fragments corresponding to the NOX1 promoter region, pulled down with a GATA3 antibody, are enriched following treatment with 50 ng/ml of IL-4 for 24 h. Chromatin immunoprecipitation assay and subsequent quantitative PCR were performed on HT-29 cells exposed to IL-4 (50 ng/ml) or solvent. Isotype-matched IgG was used as the control. Data represent the mean ± SD of three experiments. *** = P < 0.001.

Article Snippet: Human primers : NOX1 (catalog no. Hs00246589_m1), β-actin (catalog no. Hs99999903_m1), NOXA1 (catalog no. Hs00611456_g1), NOXO1 (catalog no. Hs00376039_g1), NOX2-5, DUOX1 and DUOX2 (catalog nos.

Techniques: Translocation Assay, Western Blot, Knockdown, Expressing, Transfection, Quantitative RT-PCR, Immunoprecipitation, Phospho-proteomics, Solvent, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Control

Journal: Oncotarget

Article Title: Interleukin-4 and interleukin-13 increase NADPH oxidase 1-related proliferation of human colon cancer cells

doi: 10.18632/oncotarget.17494

Figure Lengend Snippet: A . Schematic illustration of the NOX1 promoter region and its variants generated by mutagenesis, highlighting the potential GATA3 binding sites. Mutated binding sites are shown in red. These promoter regions were individually constructed as PGL3 reporters. B . Mutation at the -321/−315 region abolishes luciferase activity associated with IL-4-mediated NOX1 promoter activity. HT-29 cells were transfected with the individual reporter constructs in the presence or absence of IL-4 (50 ng/ml), and luciferase activity was measured 48 h later. Data represent the mean ± SD of three experiments. *** = P < 0.001; n.s. = not significant.

Article Snippet: Human primers : NOX1 (catalog no. Hs00246589_m1), β-actin (catalog no. Hs99999903_m1), NOXA1 (catalog no. Hs00611456_g1), NOXO1 (catalog no. Hs00376039_g1), NOX2-5, DUOX1 and DUOX2 (catalog nos.

Techniques: Generated, Mutagenesis, Binding Assay, Construct, Luciferase, Activity Assay, Transfection

Journal: Oncotarget

Article Title: Interleukin-4 and interleukin-13 increase NADPH oxidase 1-related proliferation of human colon cancer cells

doi: 10.18632/oncotarget.17494

Figure Lengend Snippet: A . Twenty pairs of human colon cancer surgical specimens and adjacent normal colonic epithelium were analyzed by quantitative RT-PCR for the expression of NOX1 and its functional isoform NOX1-L. β-actin served as the control. Paired, two-tailed t tests were used to evaluate the results; the Wilcoxon signed rank test was also used to test for differences in expression level between tumor and normal samples. The likelihood of our observations occurring if there was truly no difference in the expression between tumor and normal samples is < 0.0001. The lower panel shows the results of two representative patient samples in which cDNA was cloned from the surgical samples and subjected to PCR to examine the abundance of different NOX1 isoforms (NOX1-L and NOX1-S). B . NOX1 and IL-4α expression levels were examined by quantitative RT-PCR using the tumors (blue) and the respective adjacent normal tissues (red) of 20 colon cancer patients. β-actin served as the internal control. Pearson's correlation coefficient (r) and level of significance ( P ) using a two-tailed t test for both groups are shown in the figure.

Article Snippet: Human primers : NOX1 (catalog no. Hs00246589_m1), β-actin (catalog no. Hs99999903_m1), NOXA1 (catalog no. Hs00611456_g1), NOXO1 (catalog no. Hs00376039_g1), NOX2-5, DUOX1 and DUOX2 (catalog nos.

Techniques: Quantitative RT-PCR, Expressing, Functional Assay, Control, Two Tailed Test, Clone Assay

Journal: Oncotarget

Article Title: Interleukin-4 and interleukin-13 increase NADPH oxidase 1-related proliferation of human colon cancer cells

doi: 10.18632/oncotarget.17494

Figure Lengend Snippet: IL-4/IL-13 bind to the Type II IL-4 receptor activating the JAK1/STAT6 signaling pathway, triggering GATA3 nuclear translocation that drives NOX1 expression. GATA3 binding to the NOX1 promoter initiates transcription that is dependent on serine phosphorylation (S308). Increased expression of full-length NOX1 (NOX1-L) promotes ROS production which can inhibit PTP activity and increase cyclin D3 levels, leading to enhanced cell cycle traverse through S-phase and increased colon cancer cell proliferation. Human colon cancers, when compared to adjacent uninvolved colonic epithelia, demonstrate significantly higher levels of NOX1 and IL-4Rα mRNA, consistent with this model.

Article Snippet: Human primers : NOX1 (catalog no. Hs00246589_m1), β-actin (catalog no. Hs99999903_m1), NOXA1 (catalog no. Hs00611456_g1), NOXO1 (catalog no. Hs00376039_g1), NOX2-5, DUOX1 and DUOX2 (catalog nos.

Techniques: Translocation Assay, Expressing, Binding Assay, Phospho-proteomics, Activity Assay