Journal: Molecular Vision

Article Title: Involvement of TonEBP/NFAT5 in osmoadaptative response of human retinal pigmented epithelial cells to hyperosmolar stress

doi:

Figure Lengend Snippet: Kinetics of TonEBP expression in ARPE-19 cells exposed to hyperosmolar stress. A , B : ARPE-19 cells were incubated for 0, 1, 2, 4, 8, 12, or 24 h with iso-osmolar medium (control) or media containing the additional presence of 100 mM NaCl (Na100) or 200 mM sucrose (Su200). C : ARPE-19 cells were incubated for 4 h under iso-osmolar or hyperosmolar medium (Na100 or Su200), after which 1 µg/ml of actinomycin D (ActD) was added. Tonicity enhancer binding protein (TonEBP) mRNA levels were determined with real-time quantitative PCR (RT-qPCR) at 0, 2, 4, 6, and 8 h following the addition of ActD. A , C : TonEBP mRNA levels were measured with RT-qPCR as described in the Methods section. Data are expressed as relative TonEBP mRNA levels (in fold stimulation) to the 0 h time point set to 1. Data are the mean ± standard error of the mean (SEM; n=3) and are expressed as TonEBP mRNA levels following normalization with appropriate reference genes ( HPRT1 , B2M , ATP5B ). Data were analyzed using repeated-measures ANOVA and Dunnett’s post-hoc tests. *: p<0.05 and **p <0.01 indicate statistical significance compared to time 0 h. B : The TonEBP protein levels were determined with semiquantitative western blot analysis. β-actin was used as an internal control of protein expression. Data are representative of three independent experiments.

Article Snippet: ARPE-19 cells were incubated overnight at 4 °C with the primary antibodies against TonEBP (Novus Biologicals, Littleton, CO, dilution 1:200), followed by incubation with biotinylated anti-rabbit immunoglobulin (IgG; GE Healthcare UK Limited, Buckinghamshire, UK, dilution 1:600) and streptavidin-cyanin2 (Jackson Immunoresearch Laboratories, West Grove, PA, dilution 1:200).

Techniques: Expressing, Incubation, Control, Binding Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot

Journal: Molecular Vision

Article Title: Involvement of TonEBP/NFAT5 in osmoadaptative response of human retinal pigmented epithelial cells to hyperosmolar stress

doi:

Figure Lengend Snippet: Kinetics of TonEBP nuclear translocation in ARPE-19 cells exposed to hyperosmolar stress. ARPE-19 cells were incubated for 4, 8, or 12 h with iso-osmolar medium (control) or media containing the additional presence of 100 mM NaCl (Na100) or 200 mM sucrose (Su200). Cells were then fixed and exposed to immunofluorescent staining of tonicity enhancer binding protein (TonEBP) (in green) as described in the Methods section. A : Negative control (CTNeg) was performed in the sole presence of secondary antibodies. B : Cells were incubated under iso-osmolar conditions for 0 h (CT). C , E , G : Cells were incubated with Na100. D , F , H : Cells were incubated with Su200. Cell nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI; blue). Scale bars represent 20 µm. Pictures were taken at 40X magnification. Data are representative of three independent experiments.

Article Snippet: ARPE-19 cells were incubated overnight at 4 °C with the primary antibodies against TonEBP (Novus Biologicals, Littleton, CO, dilution 1:200), followed by incubation with biotinylated anti-rabbit immunoglobulin (IgG; GE Healthcare UK Limited, Buckinghamshire, UK, dilution 1:600) and streptavidin-cyanin2 (Jackson Immunoresearch Laboratories, West Grove, PA, dilution 1:200).

Techniques: Translocation Assay, Incubation, Control, Staining, Binding Assay, Negative Control

Journal: Molecular Vision

Article Title: Involvement of TonEBP/NFAT5 in osmoadaptative response of human retinal pigmented epithelial cells to hyperosmolar stress

doi:

Figure Lengend Snippet: Dose–response curve of hyperosmolar stress on TonEBP expression in ARPE-19 cells. ARPE-19 cells were incubated for 12 h with iso-osmolar medium (control) or media containing the additional presence of increasing concentrations of NaCl (Na25, Na50, Na100) or sucrose (Su50, Su100, Su200). A : Tonicity enhancer binding protein (TonEBP) mRNA levels measured with real-time quantitative PCR (RT-qPCR) under increasing concentrations of NaCl. B : TonEBP mRNA levels measured with RT-qPCR under increasing concentrations of sucrose. A , B : Data are expressed as relative TonEBP mRNA levels (in fold stimulation) over the iso-osmolar condition (Na0 or Su0) set to 1. The data are the mean ± standard error of the mean (SEM; n=3) following normalization with the appropriate reference genes ( HPRT1 , B2M , ATP5B ). Data were analyzed using repeated-measures ANOVA and Dunnett’s post-hoc tests. *: p<0.05 and **p <0.01 indicate statistical significance compared to the iso-osmolar medium (Na0 or Su0). C : The TonEBP protein levels were determined with semiquantitative western blot analysis. β-actin was used as an internal control of protein expression. Data are representative of three independent experiments.

Article Snippet: ARPE-19 cells were incubated overnight at 4 °C with the primary antibodies against TonEBP (Novus Biologicals, Littleton, CO, dilution 1:200), followed by incubation with biotinylated anti-rabbit immunoglobulin (IgG; GE Healthcare UK Limited, Buckinghamshire, UK, dilution 1:600) and streptavidin-cyanin2 (Jackson Immunoresearch Laboratories, West Grove, PA, dilution 1:200).

Techniques: Expressing, Incubation, Control, Binding Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot

Journal: Molecular Vision

Article Title: Involvement of TonEBP/NFAT5 in osmoadaptative response of human retinal pigmented epithelial cells to hyperosmolar stress

doi:

Figure Lengend Snippet: Dose–response curve of hyperosmolar stress on TonEBP nuclear translocation in ARPE-19 cells. ARPE-19 cells were incubated for 4 h with iso-osmolar medium (CT). Cells were incubated for 4 h in media containing the additional presence of increasing concentrations of NaCl (Na25, Na50, Na100; C , E , G ) or sucrose (Su50, Su100, Su200; D , F , H ). Negative control (CTNeg) was performed in the sole presence of secondary antibodies. Cells were then fixed and exposed to immunofluorescent staining of tonicity enhancer binding protein (TonEBP) (in green) as described in the Methods section. Cell nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI; blue). Scale bars represent 20 µm. Pictures were taken at 40X magnification. Data are representative of three independent experiments.

Article Snippet: ARPE-19 cells were incubated overnight at 4 °C with the primary antibodies against TonEBP (Novus Biologicals, Littleton, CO, dilution 1:200), followed by incubation with biotinylated anti-rabbit immunoglobulin (IgG; GE Healthcare UK Limited, Buckinghamshire, UK, dilution 1:600) and streptavidin-cyanin2 (Jackson Immunoresearch Laboratories, West Grove, PA, dilution 1:200).

Techniques: Translocation Assay, Incubation, Negative Control, Staining, Binding Assay

Journal: Molecular Vision

Article Title: Involvement of TonEBP/NFAT5 in osmoadaptative response of human retinal pigmented epithelial cells to hyperosmolar stress

doi:

Figure Lengend Snippet: Effects of DN-TonEBP on the transactivation activity of TonEBP. ARPE-19 cells were transiently transfected with either 10 µg of pSEAP-TonE plasmid and 10 µg of pcDNA3.1 plasmid (control plasmid) or with 10 µg of pSEAP-TonE plasmid and 10 µg of DN-TonEBP plasmid, before being incubated for 24 h in the absence (control) or presence of 100 mM additional NaCl (Na100). The activity of secreted embryonic alkaline phosphatase (SEAP) was measured with luminescence in the cell culture supernatant. Data are expressed as relative activity (in fold stimulation) over the iso-osmolar condition and are the mean ± standard error of the mean (SEM; n=3). Statistical analysis was performed with the conformity t test (###p<0.005) that compared the control in Na100 to the control in the iso-osmolar condition, and a paired t test (***p<0.005) was used to compare the dominant negative form of tonicity enhancer binding protein (DN-TonEBP) TonEBP in Na100 to DN-TonEBP in the iso-osmolar condition.

Article Snippet: ARPE-19 cells were incubated overnight at 4 °C with the primary antibodies against TonEBP (Novus Biologicals, Littleton, CO, dilution 1:200), followed by incubation with biotinylated anti-rabbit immunoglobulin (IgG; GE Healthcare UK Limited, Buckinghamshire, UK, dilution 1:600) and streptavidin-cyanin2 (Jackson Immunoresearch Laboratories, West Grove, PA, dilution 1:200).

Techniques: Activity Assay, Transfection, Plasmid Preparation, Control, Incubation, Cell Culture, Dominant Negative Mutation, Binding Assay

Journal: Molecular Vision

Article Title: Involvement of TonEBP/NFAT5 in osmoadaptative response of human retinal pigmented epithelial cells to hyperosmolar stress

doi:

Figure Lengend Snippet: Effects of DN-TonEBP on NaCl-induced TauT and AR expression. ARPE-19 cells transiently transfected with either H 2 O or 10 µg of DN-TonEBP were incubated for 8 h with iso-osmolar medium (control, CT) or media containing the additional presence of 100 mM NaCl (Na100). ( A ) Aldose reductase (AR) and ( B ) sodium-dependent taurine transporter (TauT) mRNA levels were measured with real-time quantitative PCR (RT-qPCR) as described in the Methods section. The data are the mean ± standard error of the mean (SEM; n=5) and are expressed as gene mRNA levels (in fold stimulation) over the iso-osmolar condition set to 1 for H 2 O and a dominant negative form of tonicity enhancer binding protein (DN-TonEBP) following normalization with the appropriate reference genes ( YWHAZ , ATP5B , MDH1 ). Statistical analysis was performed with the conformity t test (#p<0.05; ##p<0.01) that compared the Na100 condition to the iso-osmolar condition, and a paired t test (*p<0.05) as used to compare DN-TonEBP in Na100 to DN-TonEBP in the iso-osmolar condition.

Article Snippet: ARPE-19 cells were incubated overnight at 4 °C with the primary antibodies against TonEBP (Novus Biologicals, Littleton, CO, dilution 1:200), followed by incubation with biotinylated anti-rabbit immunoglobulin (IgG; GE Healthcare UK Limited, Buckinghamshire, UK, dilution 1:600) and streptavidin-cyanin2 (Jackson Immunoresearch Laboratories, West Grove, PA, dilution 1:200).

Techniques: Expressing, Transfection, Incubation, Control, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Dominant Negative Mutation, Binding Assay

Journal: Molecular Vision

Article Title: Involvement of TonEBP/NFAT5 in osmoadaptative response of human retinal pigmented epithelial cells to hyperosmolar stress

doi:

Figure Lengend Snippet: Involvement of p38 protein kinase in TonEBP activation and subsequent transactivation activity induced by hyperosmolar stress in ARPE-19 cells. Cells were preincubated for 1 h in the presence of 0.1% dimethyl sulfoxide (DMSO) or 10 µM SB203580 and then incubated for various times with iso-osmolar medium (CT; open columns) or medium containing the additional presence of 100 mM NaCl (Na100; closed columns). ( A ) Tonicity enhancer binding protein (TonEBP) translocation, ( B ) secreted embryonic alkaline phosphatase (SEAP) activity, and ( C ) quantification of aldose reductase (AR) and ( D ) sodium-dependent taurine transporter (TauT) mRNA levels were performed following 4, 24, 8, and 8 h incubation, respectively, as described in the Methods section. A : TonEBP was labeled in green, while cell nuclei were labeled in blue. Scale bars represent 20 µm. Pictures were taken at 40X magnification. Data are representative of three independent experiments. B : SEAP data are expressed as relative activity (in fold stimulation) over the control (DMSO) in Na100 and are the mean ± standard error of the mean (SEM; n=3). C , D : Data are expressed as relative gene mRNA levels (in fold stimulation) over the DMSO iso-osmolar condition set to 1. The data are the mean ± SEM (n=3) and are expressed as gene mRNA levels following normalization with the appropriate reference genes ( HPRT1 , B2M , ATP5B ). B , C , D : Statistical analysis was performed using the conformity t test (*p<0.05) that compared SB203580 Na100 with control Na100, a paired t test (#p<0.05, ###p<0.005) that compared SB203580 Na100 with SB203580 in the iso-osmolar condition, and a second paired t test that compared DMSO in the iso-osmolar condition and SB203580 in the iso-osmolar condition.

Article Snippet: ARPE-19 cells were incubated overnight at 4 °C with the primary antibodies against TonEBP (Novus Biologicals, Littleton, CO, dilution 1:200), followed by incubation with biotinylated anti-rabbit immunoglobulin (IgG; GE Healthcare UK Limited, Buckinghamshire, UK, dilution 1:600) and streptavidin-cyanin2 (Jackson Immunoresearch Laboratories, West Grove, PA, dilution 1:200).

Techniques: Activation Assay, Activity Assay, Incubation, Binding Assay, Translocation Assay, Labeling, Control

Journal: Neuro-Signals

Article Title: Nuclear factor of activated T cells 5 deficiency increases the severity of neuronal cell death in ischemic injury.

doi: 10.1159/000331899

Figure Lengend Snippet: Fig. 2. IgG extravasation, aquaporin-4 expression and cellular localization of NFAT5 in the brain after tMCAO. a Representa - tive micrographs with the IgG staining in NFAT5 +/+ (i, ii) and NFAT5 +/– (iii, iv) brain section after tMCAO. Similar IgG staining was detected in cerebral blood vessels (black arrows) in the contralateral side of the NFAT5 +/+ and NFAT5 +/– hemisphere (i and iii). IgG staining was induced in the area of ischemic core of the NFAT5 +/+ and NFAT5 +/– ipsilateral hemisphere (ii and iv). Increased IgG leakage (black arrows) in this area was detected in the NFAT5 +/– ipsilateral hemisphere (iv) compared to the NFAT5 +/+ ipsilateral hemisphere (ii). Con = Contralateral hemi- sphere; Ip = ipsilateral hemisphere. Scale bar = 100 m. n = 5. The histogram on the right shows the quantification of the relative IgG staining intensities in the brain sections; * * * p ! 0.01 Mann Whitney test. b Representative micrographs showing the immu- nostaining of aquaporin-4 (AQP-4) in NFAT5 +/+ ( i –iii) and

Article Snippet: Immunocytochemical (ICC) Analysis of Brain Tissue After tMCAO, brain samples were collected after 24 h. 7- m paraffin-embedded tMCAO-treated brain sections were fixed with 4% paraformaldehyde and stained with antibodies against NFAT5 (1: 500, a kind gift from Prof. H.M. Kown, University of Maryland), Occludin (1: 50, Santa Cruz) and AQP-4 (1: 200, Chemicon International).

Techniques: Expressing, Staining, MANN-WHITNEY

Journal: Neuro-Signals

Article Title: Nuclear factor of activated T cells 5 deficiency increases the severity of neuronal cell death in ischemic injury.

doi: 10.1159/000331899

Figure Lengend Snippet: Fig. 3. NFAT5 protein expression level and translocation of NFAT5 and the ORE activity in neurons after 3 h H/I injury. The primary cortical neurons were isolated from NFAT5 +/+ mice at embryonic day 14.5 and challenged to N or H/I for 3 h. a The ex- pression of NFAT5 level was detected by Western blot analysis. b Histogram showing the NFAT5 expression level was significant- ly increased after 3-hour H/I condition. Data are presented as mean 8 SEM. * p ! 0.05, by one-way ANOVA, n = 3–4. The primary cortical neurons were isolated from the NFAT5 +/+ mice at embryonic day 14.5 and subjected to 1-hour, 3-hour H/I or 3-hour N condition. Neurons were stained with NFAT5 antibodies

Article Snippet: Immunocytochemical (ICC) Analysis of Brain Tissue After tMCAO, brain samples were collected after 24 h. 7- m paraffin-embedded tMCAO-treated brain sections were fixed with 4% paraformaldehyde and stained with antibodies against NFAT5 (1: 500, a kind gift from Prof. H.M. Kown, University of Maryland), Occludin (1: 50, Santa Cruz) and AQP-4 (1: 200, Chemicon International).

Techniques: Expressing, Translocation Assay, Activity Assay, Isolation, Western Blot, Staining

Journal: Experimental & Molecular Medicine

Article Title: Reactive oxygen species regulate context-dependent inhibition of NFAT5 target genes

doi: 10.1038/emm.2013.61

Figure Lengend Snippet: NFAT5 differentially affects nuclear factor-κB (NF-κB) activity in macrophages in a stimulus-dependent manner. ( a and b ) Additive effects of LPS and NaCl on NFAT5 activation. LPS (5 μg ml −1 ) or heat-inactivated E. coli (3 × 10 6 CFU ml −1 ) were added to RAW 264.7 cells for 24 h in the presence or absence of NaCl (90 mℳ). NFAT5-green fluorescent protein (GFP) activity and NFAT5 translocation were determined by flow cytometry and western blot analysis, respectively. The data on the right in a show the mean±s.d. of three independent experiments. * P <0.01 versus LPS or heat-inactivated E. coli alone. ( c ) LPS- and high salt-induced increases in NFAT5 reporter activity in vivo . Matrigel with RAW 264.7 macrophages stably transfected with NFAT5-red fluorescent protein (RFP) reporter were subcutaneously implanted into mice. On day 9, LPS (10 mg kg −1 ) and hypertonic saline (11.3% HS, 25 cc kg −1 ) were injected intraperitoneally. Normal saline (0.9% NS, 25 cc kg −1 ) was injected as a control. At 16 h post injection, NFAT5-RFP activity in the Matrigel was determined using the Maestro Imaging System. ( d ) Transcriptional activity of NF-κB in NFAT5-deficient macrophages treated with LPS (5 μg ml −1 ) or NaCl (90 mℳ). An NF-κB reporter containing GFP was transiently transfected into cells, and GFP activity representing NF-κB was determined by flow cytometry (left). The bar graph on the right represents the mean±s.d. of five independent experiments. * P <0.05 versus control short hairpin RNA (shRNA)-transfected cells in the presence or absence of NaCl. The reduction in NFAT5 expression was confirmed by western blot analysis (top panel).

Article Snippet: To produce NFAT5-deficient macrophages, RAW 264.7 macrophages were seeded to 20% confluence in 12-well plates and were then transduced with NFAT5 short hairpin RNA-harboring lentiviral particles (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in the presence of polybrene (5 μg ml −1 ).

Techniques: Activity Assay, Activation Assay, Translocation Assay, Flow Cytometry, Western Blot, In Vivo, Stable Transfection, Transfection, Saline, Injection, Control, Imaging, shRNA, Expressing

Journal: Experimental & Molecular Medicine

Article Title: Reactive oxygen species regulate context-dependent inhibition of NFAT5 target genes

doi: 10.1038/emm.2013.61

Figure Lengend Snippet: Mutual suppression of NFAT5-governed gene expression by cotreatment with hypertonic stimuli and LPS. ( a ) Reduction in high salt-induced increases in AR, BGT1 and SMIT expression by pretreatment with LPS. RAW 264.7 macrophages were preconditioned with LPS (5 μg ml −1 ) for 4 h and then costimulated with NaCl (90 mℳ) for 4 h, or were stimulated first with NaCl for 4 h and then cotreated with LPS for an additional 4 h. The mRNA expression levels of AR, BGT1 and SMIT were assessed by real-time PCR. Fold inductions were calculated using the 2 −ΔΔCt method. The data are expressed as the mean±s.d. of three independent experiments. * P <0.01. ( b ) Suppression of LPS-triggered IL-6 expression by cotreatment with NaCl. IL-6 expression levels were determined by enzyme-linked immunosorbent assay (left panel) and real-time PCR analysis (right panel). * P <0.01. ( c ) Hypertonic regulation of IL-6 promoter activity. RAW 264.7 cells were stimulated with LPS in the presence or absence of NaCl (90 mℳ) for 24 h and were subsequently subjected to flow cytometry analysis of IL-6 promoter activity. * P <0.01.

Article Snippet: To produce NFAT5-deficient macrophages, RAW 264.7 macrophages were seeded to 20% confluence in 12-well plates and were then transduced with NFAT5 short hairpin RNA-harboring lentiviral particles (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in the presence of polybrene (5 μg ml −1 ).

Techniques: Gene Expression, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Activity Assay, Flow Cytometry

Journal: Experimental & Molecular Medicine

Article Title: Reactive oxygen species regulate context-dependent inhibition of NFAT5 target genes

doi: 10.1038/emm.2013.61

Figure Lengend Snippet: Mannitol induces NFAT5 activity but reduces LPS-induced IL-6 production. ( a and b ) Dose-dependent increases in NFAT5 reporter activity after treatment with mannitol. Mannitol (100, 200 and 400 mℳ) was added to RAW 264.7 macrophages harboring the NFAT5 reporter gene for 24 h. The bar graph in b shows the mean±s.d. of three independent experiments. ( c ) Suppression of LPS-triggered IL-6 production by cotreatment with mannitol. RAW 264.7 macrophages were stimulated with LPS (5 μg ml −1 ) for 24 h in the presence of mannitol. IL-6 levels in the supernatant were determined by enzyme-linked immunosorbent assay. * P <0.01. The degree of cell viability was assessed by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and presented as the percentage of the cell viability of treated samples to the viability of control untreated cells.

Article Snippet: To produce NFAT5-deficient macrophages, RAW 264.7 macrophages were seeded to 20% confluence in 12-well plates and were then transduced with NFAT5 short hairpin RNA-harboring lentiviral particles (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in the presence of polybrene (5 μg ml −1 ).

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: Experimental & Molecular Medicine

Article Title: Reactive oxygen species regulate context-dependent inhibition of NFAT5 target genes

doi: 10.1038/emm.2013.61

Figure Lengend Snippet: ROS regulate NFAT5 activity in a context-dependent manner. ( a ) Time kinetics of ROS production by RAW 264.7 macrophages stimulated with LPS or NaCl. ROS expression was determined by flow cytometry using an ROS probe (DCFH-DA (2',7'-dichlorofluorescein diacetate)). A representative histogram is shown on the left. The data on the right show the mean±s.d. of three independent experiments and are expressed as normalized mean fluorescence intensity (MFI) relative to the MFI determined 24 h after stimulation with 5 μg ml −1 LPS (set as 100%). * P <0.01 versus NaCl stimulation (90 mℳ). ( b ) Effects of LPS on high NaCl-induced ROS production by macrophages. ROS levels were determined 6 h after stimulation with 5 μg ml −1 LPS in the presence or absence of 90 mℳ NaCl. ( c ) Suppression of high salt-induced NFAT5 activity by myxothiazol (MTZ), a mitochondrial ROS inhibitor, but not by allopurinol (Allo). ROS scavengers, including Allo (1 mℳ) and MTZ (10 μℳ), were added to RAW 264.7 macrophages 1 h before stimulation with NaCl (90 mℳ) or LPS (5 μg ml −1 ). NFAT5-dependent reporter activity was then determined by flow cytometry. Representative histograms are shown on the left. The bar graph shows the mean±s.d. of three independent experiments.

Article Snippet: To produce NFAT5-deficient macrophages, RAW 264.7 macrophages were seeded to 20% confluence in 12-well plates and were then transduced with NFAT5 short hairpin RNA-harboring lentiviral particles (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in the presence of polybrene (5 μg ml −1 ).

Techniques: Activity Assay, Expressing, Flow Cytometry, Fluorescence

Journal: Experimental & Molecular Medicine

Article Title: Reactive oxygen species regulate context-dependent inhibition of NFAT5 target genes

doi: 10.1038/emm.2013.61

Figure Lengend Snippet: ROS control LPS- and high salt-induced suppression of NFAT5 target genes. ( a and b ) ROS dependency of NFAT5 target gene expression induced by LPS or high salt. The mRNA expression of AR, SMIT, IL-6 and NOS2 was determined by real-time PCR analysis 4 h after stimulation with NaCl or LPS. The data are the mean±s.d. of three independent experiments. * P <0.05 versus NaCl (A) or LPS alone (B). ( c and d ) ROS inhibitors restore SMIT, IL-6 and MCP-1 expression after combined treatment with high salt and LPS. RAW 264.7 macrophages were pretreated with various concentrations of allopurinol (Allo), rotenone (Ro), or myxothiazol (MTZ) for 1 h and then stimulated with LPS (5 μg ml −1 ) and NaCl (90 mℳ) for 4 h. IL-6 and MCP-1 concentrations in the culture supernatants were assessed by enzyme-linked immunosorbent assay. SMIT mRNA expression was determined by real-time PCR. * P <0.01. The degree of cell viability was assessed by an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and presented as the percentage of the cell viability of treated samples to the viability of control untreated cells.

Article Snippet: To produce NFAT5-deficient macrophages, RAW 264.7 macrophages were seeded to 20% confluence in 12-well plates and were then transduced with NFAT5 short hairpin RNA-harboring lentiviral particles (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in the presence of polybrene (5 μg ml −1 ).

Techniques: Control, Targeted Gene Expression, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Experimental & Molecular Medicine

Article Title: Reactive oxygen species regulate context-dependent inhibition of NFAT5 target genes

doi: 10.1038/emm.2013.61

Figure Lengend Snippet: Rotenone restores the high salt-induced suppression of NFAT5 binding to the IL-6 promoter. ( a ) Chromatin immunoprecipitation (ChIP) assay for the NFAT5 binding site within the IL-6 promoter. RAW 264.7 macrophages were stimulated with LPS (5 μg ml −1 ) for 10 h. DNA isolated from anti-NFAT5 precipitation complexes was amplified by PCR using specific primers for the promoter or exon regions of IL-6. The exon region of the IL-6 gene was used as a negative control. ( b ) RAW 264.7 macrophages were pretreated with allopurinol (Allo; 1 mℳ) or rotenone (Ro; 10 μℳ) for 1 h, stimulated with LPS (5 μg ml −1 ) and NaCl (90 mℳ) for 10 h, and then subjected to the ChIP assay. The PCR band densities were normalized relative to the input signals and quantified using ImageJ software (top panel). A representative of three independent experiments is shown. IP, immunoprecipitation.

Article Snippet: To produce NFAT5-deficient macrophages, RAW 264.7 macrophages were seeded to 20% confluence in 12-well plates and were then transduced with NFAT5 short hairpin RNA-harboring lentiviral particles (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in the presence of polybrene (5 μg ml −1 ).

Techniques: Binding Assay, Chromatin Immunoprecipitation, Isolation, Amplification, Negative Control, Software, Immunoprecipitation

Journal: Experimental & Molecular Medicine

Article Title: Reactive oxygen species regulate context-dependent inhibition of NFAT5 target genes

doi: 10.1038/emm.2013.61

Figure Lengend Snippet: In vivo evidence for the suppression of NFAT5-governed genes by cotreatment with high salt and LPS. ( a ) High salt-induced suppression of IL-6 mRNA expression in the spleen. LPS (10 mg kg −1 ) was injected intraperitoneally into mice for 7 h after challenging the animals with hypertonic saline (HS; 11.3% HS, 25 cc kg −1 ) or normal saline (NS; 0.9% NS, 25 cc kg −1 ). mRNA expression of IL-6 in the spleen was analyzed by real-time PCR. * P <0.01. ( b and c ) LPS-mediated regulation of tonicity-responsive genes in the kidney. Under the same conditions as in a , the mRNA expression of IL-6, SMIT and AR in the kidney cells was analyzed by real-time PCR. * P <0.01. ( d ) Hypothetical model depicting the role of xanthine oxidase-derived ROS in the context-dependent activation of NFAT5, leading to IL-6 production upon TLR-4 ligation. ROS (XO), xanthine oxidase-derived ROS; ROS (M), mitochondrially generated ROS.

Article Snippet: To produce NFAT5-deficient macrophages, RAW 264.7 macrophages were seeded to 20% confluence in 12-well plates and were then transduced with NFAT5 short hairpin RNA-harboring lentiviral particles (Santa Cruz Biotechnology, Santa Cruz, CA, USA) in the presence of polybrene (5 μg ml −1 ).

Techniques: In Vivo, Expressing, Injection, Saline, Real-time Polymerase Chain Reaction, Derivative Assay, Activation Assay, Ligation, Generated

Journal: American Journal of Physiology - Renal Physiology

Article Title: Identification of NFAT5 as a transcriptional regulator of the EDN1 gene in collecting duct

doi: 10.1152/ajprenal.00509.2018

Figure Lengend Snippet: Chromatin immunoprecipitation assay using an NFAT5 antibody in inner medullary collecting duct 3 cells exposed to 300 or 450 mosM (using mannitol) for 4 h. A is a representative gel showing PCR products of DNA pulled down by the NFAT5 antibody using primers (numbered 1–6) flanking regions in the mouse endothelin-1 (ET-1) promoter as indicated by the bars in B and as described in Table 1. Highlighted regions in the ET-1 promoter (B) indicate consensus NFAT5 binding sites. Bold font in B indicates exon 1. C: quantitative PCR results for the 6 primers. IgG, nonspecific IgG used as a control.

Article Snippet: For determination of mRNA levels in NFAT5-deficient cells, two different NFAT5 primers were used that amplified across the region encoded by exon 4 in the NFAT5 gene (TaqMan Mm00957045_g1 and Mm01247392_m1). siRNA Studies Mouse NFAT5 siRNA and negative controls (scrambled siRNA sequences) were purchased from Origene (Rockville, MD).

Techniques: Chromatin Immunoprecipitation, Binding Assay, Real-time Polymerase Chain Reaction, Control

Journal: American Journal of Physiology - Renal Physiology

Article Title: Identification of NFAT5 as a transcriptional regulator of the EDN1 gene in collecting duct

doi: 10.1152/ajprenal.00509.2018

Figure Lengend Snippet: Effect of increasing media osmolarity for 30 min or 2 h (300 vs. 450 mosM using mannitol) on NFAT5 cytoplasmic and nuclear protein content. The cytoplasmic fraction Western blot and densitometry are shown in A and C, respectively; the nuclear fraction Western blot and densitometry and shown in B and D, respectively. n = 3 for each data point, except n = 2 for nuclear fraction 2 h. *P < 0.05 vs. 300 mosM. TBP, TATA-binding protein.

Article Snippet: For determination of mRNA levels in NFAT5-deficient cells, two different NFAT5 primers were used that amplified across the region encoded by exon 4 in the NFAT5 gene (TaqMan Mm00957045_g1 and Mm01247392_m1). siRNA Studies Mouse NFAT5 siRNA and negative controls (scrambled siRNA sequences) were purchased from Origene (Rockville, MD).

Techniques: Western Blot, Binding Assay

Journal: American Journal of Physiology - Renal Physiology

Article Title: Identification of NFAT5 as a transcriptional regulator of the EDN1 gene in collecting duct

doi: 10.1152/ajprenal.00509.2018

Figure Lengend Snippet: Effect of NFAT5 small-interfering RNA (siRNA) on endothelin-1 (ET-1, A) and NFAT5 (B) mRNA content [normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA content] after exposure to 300 or 450 mosM (using mannitol) for 2 h. Scrambled siRNA was used as a control. n = 6 for each data point. *P < 0.05 vs. 300 mosM under the same conditions (scrambled or NFAT5 siRNA); #P < 0.05 vs. 450 mosM scrambled siRNA.

Article Snippet: For determination of mRNA levels in NFAT5-deficient cells, two different NFAT5 primers were used that amplified across the region encoded by exon 4 in the NFAT5 gene (TaqMan Mm00957045_g1 and Mm01247392_m1). siRNA Studies Mouse NFAT5 siRNA and negative controls (scrambled siRNA sequences) were purchased from Origene (Rockville, MD).

Techniques: Small Interfering RNA, Control

Journal: American Journal of Physiology - Renal Physiology

Article Title: Identification of NFAT5 as a transcriptional regulator of the EDN1 gene in collecting duct

doi: 10.1152/ajprenal.00509.2018

Figure Lengend Snippet: Effect of increasing media osmolarity (using mannitol) for 2 h on endothelin-1 (ET-1) mRNA content [normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA content] in wild-type inner medullary collecting duct (IMCD) 3 cells or in IMCD3 cells with CRISPR/Cas-mediated targeted disruption of exon 4 in the NFAT5 gene. n = 6 for each data point. *P < 0.05 vs. 300 mosM in wild-type IMCD3 cells; #P < 0.05 vs. 450 mosM in wild-type IMCD3 cells.

Article Snippet: For determination of mRNA levels in NFAT5-deficient cells, two different NFAT5 primers were used that amplified across the region encoded by exon 4 in the NFAT5 gene (TaqMan Mm00957045_g1 and Mm01247392_m1). siRNA Studies Mouse NFAT5 siRNA and negative controls (scrambled siRNA sequences) were purchased from Origene (Rockville, MD).

Techniques: CRISPR, Disruption

Journal: American Journal of Physiology - Renal Physiology

Article Title: Identification of NFAT5 as a transcriptional regulator of the EDN1 gene in collecting duct

doi: 10.1152/ajprenal.00509.2018

Figure Lengend Snippet: Effect of osmolarity on endothelin-1 (ET-1) promoter activity (assessed by the ratio of secreted luciferase/secreted alkaline phosphatase) in inner medullary collecting duct 3 cells. In A, cells were transfected with 1-, 2-, or 3-kb ET-1 promoter-reporter constructs and then exposed to 300 or 350 mosM (using mannitol) for varying time periods. In B, cells were transfected with a wild-type 1-kb ET-1 promoter-reporter construct or a 1-kb ET-1 promoter construct in which the two NFAT5 binding sites were mutated and then exposed to 300 or 350 mosM (using mannitol) for 4 h. n = 6–9 for each data point. *P < 0.05 vs. 300 mosM at the same time point; #P < 0.05 vs. wild-type 1-kb promoter at 300 mosM.

Article Snippet: For determination of mRNA levels in NFAT5-deficient cells, two different NFAT5 primers were used that amplified across the region encoded by exon 4 in the NFAT5 gene (TaqMan Mm00957045_g1 and Mm01247392_m1). siRNA Studies Mouse NFAT5 siRNA and negative controls (scrambled siRNA sequences) were purchased from Origene (Rockville, MD).

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

Journal: American Journal of Physiology - Renal Physiology

Article Title: Identification of NFAT5 as a transcriptional regulator of the EDN1 gene in collecting duct

doi: 10.1152/ajprenal.00509.2018

Figure Lengend Snippet: Effect of osmolarity on endothelin-1 (ET-1) promoter activity (assessed by the ratio of secreted luciferase/secreted alkaline phosphatase) in wild-type inner medullary collecting duct (IMCD) 3 cells or in IMCD3 cells with CRISPR/Cas-mediated targeted disruption of exon 4 in the NFAT5 gene. Cells were transfected with the 1-kb ET-1 promoter-reporter construct and exposed to 300 or 350 mosM (using mannitol) for 4 h. n = 9 for each data point. *P < 0.05 vs. wild-type IMCD3.

Article Snippet: For determination of mRNA levels in NFAT5-deficient cells, two different NFAT5 primers were used that amplified across the region encoded by exon 4 in the NFAT5 gene (TaqMan Mm00957045_g1 and Mm01247392_m1). siRNA Studies Mouse NFAT5 siRNA and negative controls (scrambled siRNA sequences) were purchased from Origene (Rockville, MD).

Techniques: Activity Assay, Luciferase, CRISPR, Disruption, Transfection, Construct

Journal: Nature Communications

Article Title: Tissue sodium excess is not hypertonic and reflects extracellular volume expansion

doi: 10.1038/s41467-020-17820-2

Figure Lengend Snippet: Panel a bars: empty = WKY , dashed = SHRSP , red = high salt (HS) treatment; all bars show mean ± 95% CI, with individual points (overlay; + and x: female and male automatically detected outliers, respectively; ROUT, Q = 1%); significant differences at predefined comparisons are shown by brackets, on top (two-sided Fisher LSD test; please see also Supplementary Table for sex-specific data and detailed significance). Tissue Na + content increased in all tissues in SHRSP -HS, but in myocardium; with the exception of skeletal muscle, it was consistently paralleled by water accrual. Total Na + + K + concentration in tissues fell in physiological ranges (light blue) and was unaffected by salt loading in either strain, ruling out hypertonic accumulations. Panel b data presented as Delta Ct, median ± 95% CI for all rats; significant differences at two-sided Mann–Whitney test are shown in brackets. Increased TonEBP gene expression followed Na + and water accumulation despite no hypertonicity. For both panels, n = 16–18 animals/group, as per Supplementary Data ; source data are provided as a Source Data file.

Article Snippet: For qRT-PCR, CDNA was prepared using the High capacity CDNA reverse transcription kit (Applied Biosystems) and analysed using Taqman fast advanced master mix with specific Taqman gene expression assay probes for TonEBP (Rn01762487_m1), GAPDH (Rn01462661 g1), beta-actin (Rn00667869 m1).

Techniques: Concentration Assay, MANN-WHITNEY, Gene Expression