Journal: The FASEB Journal

Article Title: Loss of Nfat5 promotes lipid accumulation in vascular smooth muscle cells

doi: 10.1096/fj.202100682r

Figure Lengend Snippet: FIGURE 2 Analysis of the responses of human VSMCs to cholesterol stimulation. Human VSMCs were treated with cholesterol- supplemented medium (Panserin, chol:MbCD 10 µg/mL) for three days. The expression of genes encoding determinants of cholesterol and phospholipid processing was analyzed by semi-quantitative PCR (A, **P < .01, *P < .05, n = 6, the expression of the housekeeping gene RPL32 served as internal standard). NFAT5 was detected by immunostaining. NFAT5-positive nuclei were detected by immunofluorescence-based methods and quantified by automated image analysis (B, ***P < .001, n = 10, scale bar: 50 µm). NFAT5 protein abundance was determined in nuclear and cytosolic fractions of cell lysates by immunoblot techniques (C and D, *P < .05, n = 6, histone H3 and α-tubulin served as loading controls for the nuclear and cytosolic fractions, respectively. Ctr., control; Chol., cholesterol)

Article Snippet: Cells were incubated with a rabbit anti- NFAT5 antibody (NB120- 3446; Novus Biologicals) at 4°C overnight.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Immunostaining, Immunofluorescence, Quantitative Proteomics, Western Blot, Control

Journal: The FASEB Journal

Article Title: Loss of Nfat5 promotes lipid accumulation in vascular smooth muscle cells

doi: 10.1096/fj.202100682r

Figure Lengend Snippet: FIGURE 3 Analysis of the responses of mouse aortic VSMCs to cholesterol stimulation. VSMCs isolated from aortae of Nfat5fl/fl mice were exposed to cholesterol-supplemented medium (Panserin, chol:MbCD 10 µg/mL) or the respective solvent control for three days. Abca1, Acat1, Hmgcr, Lgals3, and Cd68 expression was analyzed by real-time quantitative PCR (A). The expression of the housekeeping gene Rps12 served as internal control (A, *P < .05, ***P < .001 vs control, n = 6-7). NFAT5 protein was detected by capillary electrophoresis in cytosolic and nuclear fractions of cell lysates B and C. HDAC1 and α-tubulin served as loading controls for the nuclear and cytosolic fractions respectively (C, Cytosol: n.s. vs control; Nucleus: *P < .05 vs control, n = 3, Ctr., control; Chol., cholesterol; n.s., not significant)

Article Snippet: Cells were incubated with a rabbit anti- NFAT5 antibody (NB120- 3446; Novus Biologicals) at 4°C overnight.

Techniques: Isolation, Solvent, Control, Expressing, Real-time Polymerase Chain Reaction, Electrophoresis

Journal: The FASEB Journal

Article Title: Loss of Nfat5 promotes lipid accumulation in vascular smooth muscle cells

doi: 10.1096/fj.202100682r

Figure Lengend Snippet: FIGURE 4 Genetic ablation of Nfat5 and its effect on cholesterol-exposed VSMCs. Genetic ablation of Nfat5 was achieved by expressing Cre-recombinase in murine Nfat5fl/fl VSMCs via adenoviral transduction (AdCre, an empty vector adenovirus (AdPl) served as control). The knockout of Nfat5 was evidenced by real-time quantitative PCR analysis (A, ***P < .001 vs control, n = 6, Rps12 expression level served as reference) and confirmed on protein level (B, results from capillary electrophoresis and automated signal detection, Valosin-containing protein (VCP) served as loading reference). VSMCs were exposed to cholesterol- supplemented medium for three days (Panserin, chol:MbCD 10 µg/ mL) 24 h after viral transduction which did not alter the expression of genes associated with a foam-cell like phenotype (C, n.s. vs control, n = 6, Chol., cholesterol; n.s., not significant)

Article Snippet: Cells were incubated with a rabbit anti- NFAT5 antibody (NB120- 3446; Novus Biologicals) at 4°C overnight.

Techniques: Expressing, Transduction, Plasmid Preparation, Control, Knock-Out, Real-time Polymerase Chain Reaction, Electrophoresis

Journal: The FASEB Journal

Article Title: Loss of Nfat5 promotes lipid accumulation in vascular smooth muscle cells

doi: 10.1096/fj.202100682r

Figure Lengend Snippet: FIGURE 5 Identification of Nfat5-controlled arteriosclerosis-associated transcriptional targets. The Nfat5-regulated expression of selected transcripts associated with the development of arteriosclerotic plaques was analyzed by applying a macroarray-based PCR screening protocol comparing cholesterol-exposed murine Nfat5fl/fl and Nfat5−/− VSMCs. Transcripts which were at least four-fold (rounded up) down- regulated in Nfat5-deficient VSMCs were summarized (A, significantly regulated transcripts were marked in yellow and their individual results were shown in B, *P < .05 vs control, n = 3, Ctr., control; Chol., cholesterol; Hsp90ab1 served as reference; comparable results were obtained if Gapdh was chosen as reference). The functional effect of the Nfat5 knockout was analyzed by comparing the cholesterol accumulation in cholesterol-exposed murine Nfat5fl/fl and Nfat5−/− VSMCs (C, Oil Red O (ORO)-stained area, **P < .01 vs. control, one out of two independent experiments with comparable results analyzing 37-62 cells per group is shown, scale bar: 50 µm). Additionally, the cellular cholesterol content was assessed by utilizing a direct colorimetric cholesterol assay (D, *P < .05 vs control, n = 4). Lipids were also visualized in murine aortic VSMCs overexpressing NFAT5 (AdN5; control: AdPl) after exposure to cholesterol (E, ***P < .001 vs. control, one out of three independent experiments with comparable results determining the ORO-positive area of 37-39 cells, scale bars: 50 µm)

Article Snippet: Cells were incubated with a rabbit anti- NFAT5 antibody (NB120- 3446; Novus Biologicals) at 4°C overnight.

Techniques: Expressing, Control, Functional Assay, Knock-Out, Staining, Cholesterol Assay

Journal: The FASEB Journal

Article Title: Loss of Nfat5 promotes lipid accumulation in vascular smooth muscle cells

doi: 10.1096/fj.202100682r

Figure Lengend Snippet: FIGURE 6 Analyses of blood cholesterol levels and lipid accumulation in the aorta of mice fed an atherogenic diet upon SMC-specific genetic ablation of Nfat5. Nfat5fl/fl (N5fl/fl) and Nfat5(SMC)−/− (N5(SMC)−/−) mice were fed an atherogenic diet (AD) for either 14 or 25 weeks or a normal chow diet (ND). Blood cholesterol levels were significantly elevated in mice fed an atherogenic diet (A, *P < .05 vs N5fl/fl ND, n = 4-8; **P < .01 vs N5(SMC)−/− ND, n = 6-9). Aortae were excised and stained with Oil Red O (ORO) to detect lipid-containing areas (B, scale bars: 1 mm). The cumulative ORO-stained vessel area was recorded via light microscopy and quantified (C, ***P < .001, **P < .01, *P < .05 vs control, n = 5-9, representative images are shown in D) and the location of ORO-positive areas in Nfat5(SMC)−/− mice was mapped (E). Confocal microscopy-based analyses of whole mount preparations of these aortae revealed ORO-positive droplets in the subintimal layers (F, scale bars: 100 and 40 µm)

Article Snippet: Cells were incubated with a rabbit anti- NFAT5 antibody (NB120- 3446; Novus Biologicals) at 4°C overnight.

Techniques: Staining, Light Microscopy, Control, Confocal Microscopy

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: Nature Communications

Article Title: Dehydration promotes intracellular lipid synthesis and accumulation

doi: 10.1038/s41467-026-73534-x

Figure Lengend Snippet: A Immunoblots of total Acetyl-CoA Carboxylase (ACC), pACC-S79/221, and NFAT5 in CD cells transfected with si Nfat5 vs siCtrl treated with NaCl or control n = independent biologic replicates. B Immunofluorescence of BODIPY-stained lipid droplets in CD cells treated with siCtrl or si Nfat5 +/− NaCl n = images analyzed from three independent experiments. C Cleavage Under targets & Tagmentation (CUT&TAG) of kidney collecting duct (CD) cells under isotonic and hypertonic conditions, showing NFAT5 occupancy in the promoter region of genes associated to lipid synthesis and/or elongation, H3K27ac (positive control), and H3K27me3 (negative control). y-axis denotes reads per genomic content (RPGC). D Urine osmolality, kidney lysate triglycerides, and Nfat5 mRNA in NFAT5 heterozygous KO mice vs controls after water restriction. E Net enrichment score for gene sets associated to lipid synthesis in NFAT5 kidney tubule KO mice vs WT animals after pairwise comparison analysis. Data in ( A , B ) presented as mean +/− SD, analyzed with two-way ANOVA with NaCl and siRNA as the independent variables. P < 0.0001for the interaction, n as noted in figure. Data in ( D ) presented as mean +/− SD analyzed with a two-tailed Student’s t test. n as indicated in each panel. CD – Kidney collecting duct cells. Source data are provided as a Source Data file. https://BioRender.com/1vm1hyd .

Article Snippet: 1 μg of NFAT5 (Bethyl, Rb pAb, A305-174A), H3K27ac (Active motif, Ms mAb, 39685), or H3K27me3 (Active motif, Rb pAb, 39155) antibodies were added to each reaction and incubated overnight with orbital mixing at 4 o C. Reactions were washed with Digitonin150 buffer (final concentrations: Wash150 buffer, 100xPI, 0.05% digitonin) three times.

Techniques: Western Blot, Transfection, Control, Immunofluorescence, Staining, Positive Control, Negative Control, Comparison, Two Tailed Test

Journal: Cell Death & Disease

Article Title: TonEBP modulates the protective effect of taurine in ischemia-induced cytotoxicity in cardiomyocytes

doi: 10.1038/cddis.2015.372

Figure Lengend Snippet: Taurine could attenuate the enhanced proteasomal degradation of TonEBP under ischemia both in vivo and in vitro . The ischemia could promote the posttranscriptional degradation of TonEBP, as the mRNA level of TonEBP did not change in vitro or in vivo ( a , N =4); the protein level, however, decreased significantly ( b and c , N =4). The supplementation of taurine could not affect the expression level of TonEBP mRNA ( a ), but could ameliorate the ischemia-induced degradation of TonEBP ( b and c ). The following mechanism study supported proteasomal degradation involved under ischemic insults ( d – f ), as the protein degraded gradually with protein synthesis inhibited by CHX ( d , N =3), and the proteasome inhibitor, MG-132, could inhibit such degradation ( e , N =3). Furthermore, taurine could inhibit the activation of ubiquitin-proteasomal degradation of TonEBP ( f , N =3). Therefore, we confirm that taurine could attenuate the posttranscriptional degradation of TonEBP by inhibiting the ubiquitin-proteasomal pathway. Staining for RFL-32 was performed to indicate approximate equal loading of samples. (Data were expressed as mean±S.E.M. ** P <0.01; CHX, cycloheximide; CTL, control; NS, no significance; LAD, left anterior descending coronary artery ligation; OGD, oxygen glucose deprivation; Tau, taurine)

Article Snippet: HCMs were transfected with either TauT shRNA(h) plasmid (catalog no. sc-61648-SH, Santa Cruz, CA, USA) to block the expression of TauT or with TonEBP shRNA(h) plasmid (catalog no. sc-43968-SH, Santa Cruz) to block the expression of TonEBP using HilyMax Transfection Reagent according to the manufacturer's protocol.

Techniques: In Vivo, In Vitro, Expressing, Activation Assay, Ubiquitin Proteomics, Staining, Control, Ligation

Journal: Cell Death & Disease

Article Title: TonEBP modulates the protective effect of taurine in ischemia-induced cytotoxicity in cardiomyocytes

doi: 10.1038/cddis.2015.372

Figure Lengend Snippet: Taurine could promote the translocation of TonEBP to the nucleus. In our study, taurine could promote the nuclear localization of TonEBP as detected by immunofluorescence ( a ), decreasing the cytosolic TonEBP and increasing the nuclear TonEBP both in vivo and in vitro ( b and c , N =3). The results were similar in the normal condition and under ischemia. Staining for RFL-32 was performed to indicate approximate equal loading of samples. Staining for TFIIB was performed to indicate approximate equal loading of nuclear samples. (Data were expressed as mean±S.E.M. ** P <0.01; LAD, left anterior descending coronary artery ligation; OGD, oxygen glucose deprivation)

Article Snippet: HCMs were transfected with either TauT shRNA(h) plasmid (catalog no. sc-61648-SH, Santa Cruz, CA, USA) to block the expression of TauT or with TonEBP shRNA(h) plasmid (catalog no. sc-43968-SH, Santa Cruz) to block the expression of TonEBP using HilyMax Transfection Reagent according to the manufacturer's protocol.

Techniques: Translocation Assay, Immunofluorescence, In Vivo, In Vitro, Staining, Ligation

Journal: Cell Death & Disease

Article Title: TonEBP modulates the protective effect of taurine in ischemia-induced cytotoxicity in cardiomyocytes

doi: 10.1038/cddis.2015.372

Figure Lengend Snippet: Taurine could promote the transcriptional activity of TauT by TonEBP-TonE occupancy in the TauT promoter region. ( a ) Analysis of TauT promoter showed a conserved TonE-binding sites at −110 bases and −109 bases from the transcription start site. ( b ) In the ChIP assay, the TonEBP-TonE occupancy decreased significantly under ischemic insults, and taurine could increase the binding of TonEBP with TonE significantly both in vivo and in vitro ( N =6). ( c ) The proximal site of TauT promoter region (−99 to −124 bp) functions as an efficient cis -element of TonEBP, in the ischemia and taurine-modulated regulation of TauT ( N =4). The luciferase activities in cells transfected with pTauT/-124-Luc decreased significantly under ischemia, whereas taurine strongly attenuated such reduction. In the cells transfected by the pTauT/-99-Luc or pTauT/-124mut-Luc reporter, the luciferase activities decreased to an extremely low level. The luciferase activities (data were expressed as mean±S.E.M. ** P <0.01; ## P <0.01 versus the OGD group in c ; NS, no significance versus the control group; CTL, control group)

Article Snippet: HCMs were transfected with either TauT shRNA(h) plasmid (catalog no. sc-61648-SH, Santa Cruz, CA, USA) to block the expression of TauT or with TonEBP shRNA(h) plasmid (catalog no. sc-43968-SH, Santa Cruz) to block the expression of TonEBP using HilyMax Transfection Reagent according to the manufacturer's protocol.

Techniques: Activity Assay, Binding Assay, In Vivo, In Vitro, Luciferase, Transfection, Control

Journal: Cell Death & Disease

Article Title: TonEBP modulates the protective effect of taurine in ischemia-induced cytotoxicity in cardiomyocytes

doi: 10.1038/cddis.2015.372

Figure Lengend Snippet: TonEBP could regulate TauT expression and taurine content in HCMs. In our study, TonEBP overexpression could upregulate TauT expression ( a , N =3), increase the intracellular taurine contents ( b , N =4) with or without ischemic insults. The similar results could also be confirmed in the condition of TauT knockdown ( c , N =4). TonEBP knockdown could decrease TauT expression ( d , N =3), further decrease the taurine levels, and even exogenous taurine supplementation could not upregulate taurine content ( e , N =4). Staining for RFL-32 was performed to indicate approximate equal loading of samples. (Data were expressed as mean±S.E.M. * P <0.05; ** P <0.01; NS, no significance; OGD, oxygen glucose deprivation)

Article Snippet: HCMs were transfected with either TauT shRNA(h) plasmid (catalog no. sc-61648-SH, Santa Cruz, CA, USA) to block the expression of TauT or with TonEBP shRNA(h) plasmid (catalog no. sc-43968-SH, Santa Cruz) to block the expression of TonEBP using HilyMax Transfection Reagent according to the manufacturer's protocol.

Techniques: Expressing, Over Expression, Knockdown, Staining

Journal: Cell Death & Disease

Article Title: TonEBP modulates the protective effect of taurine in ischemia-induced cytotoxicity in cardiomyocytes

doi: 10.1038/cddis.2015.372

Figure Lengend Snippet: TonEBP exhibited anti-apoptotic and anti-oxidative effects in HCMs under ischemia. TonEBP overexpression significantly increased the ratio of cell viability ( a , N =3), decreased cell injury ( b , N =3) and attenuated DNA fragmentation ( c , N =3) in HCMs under the condition of OGD. Similarly, the TonEBP overexpression could attenuate the increased ROS ( d , N =3) and calcium content ( e , N =3) and ameliorate the depletion of ATP content ( f , N =3) in OGD HCMs. Also, TonEBP overexpression could significantly reduce caspase-3 activity ( g , N =3), inhibit the release of cytochrome c ( h and i , N =3) and decrease the ratio of Bax/Bcl-2 ( h , j , N =6) in HCMs under ischemic insults. Staining for RFL-32 was performed to indicate approximate equal loading of samples. Staining for COX IV was performed to indicate approximate equal loading of mitochondrial samples. (Data were expressed as mean±S.E.M. * P <0.05; ** P <0.01; NS, no significance; OE, overexpression; OGD, oxygen glucose deprivation; Tau, taurine; Vec, vector)

Article Snippet: HCMs were transfected with either TauT shRNA(h) plasmid (catalog no. sc-61648-SH, Santa Cruz, CA, USA) to block the expression of TauT or with TonEBP shRNA(h) plasmid (catalog no. sc-43968-SH, Santa Cruz) to block the expression of TonEBP using HilyMax Transfection Reagent according to the manufacturer's protocol.

Techniques: Over Expression, Activity Assay, Staining, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: TonEBP modulates the protective effect of taurine in ischemia-induced cytotoxicity in cardiomyocytes

doi: 10.1038/cddis.2015.372

Figure Lengend Snippet: The protective effects of taurine on ischemic HCMs are TonEBP dependent. The knockdown of TonEBP decreased the ratio of cell viability ( a , N =3), increased the LDH release level ( b , N =3), increased the DNA fragmentation ( c , N =3) and Annexin V-FITC and PI staining ( d , N =3). Taurine supplementation could decrease the ratio of cell death and apoptosis ( d , N =3). In the oxidative stress tests, TonEBP knockdown could exacerbate ROS production ( e , N =3), calcium overload ( f , N =3) and ATP depletion ( g , N =3), associated with increased levels of caspase-3 ( h , N =3). Similarly, both the Bax/Bcl-2 ratio ( i and j , N =3) and cytochrome c release ( i , k , N =6) were enhanced in TonEBP knockdown groups. With TonEBP knockdown, the further treatment of taurine no longer reverse the myocardial injury caused by OGD ( b – h ). (Data were expressed as mean±S.E.M. * P <0.05; ** P <0.01; NS, no significance; OGD, oxygen glucose deprivation; Tau, taurine; Vec, vector)

Article Snippet: HCMs were transfected with either TauT shRNA(h) plasmid (catalog no. sc-61648-SH, Santa Cruz, CA, USA) to block the expression of TauT or with TonEBP shRNA(h) plasmid (catalog no. sc-43968-SH, Santa Cruz) to block the expression of TonEBP using HilyMax Transfection Reagent according to the manufacturer's protocol.

Techniques: Knockdown, Staining, Plasmid Preparation

Journal: Molecular medicine reports

Article Title: Elevation of hypertonicity‑induced protein NFAT5 promotes apoptosis of human umbilical vein endothelial cells through the NF‑κB pathway.

doi: 10.3892/mmr.2021.11823

Figure Lengend Snippet: Figure 1. Hypertonic culture medium causes cell death of HUVECs and induces caspase‑3 cleavage. (A) AAA tissue samples and adjacent healthy aorta abdominalis Ctr were collected from patients and subjected to RT‑qPCR to detect the mRNA levels of NFAT5. Data were normalized to GAPDH mRNA levels. All RT‑qPCR experiments were performed in triplicate and the results were presented as the fold change relative to Ctr. **P<0.01, paired t‑test. HUVECs were cultured in isotonic or hypertonic medium. After 8 h of incubation, the cells were subjected to (B) phase contrast microscopy (scale bar, 200 µm), (C) MTS cell viability assay and (D) western blotting detection of NFAT5 and cleaved caspase‑3. β‑actin was used as a loading control. Cell viability is presented as a percentage of the Ctr. n=9 in each group. **P<0.01, Student's t‑test. HUVEC, human umbilical vein endothelial cell; AAA, abdominal aortic aneurysm; Ctr, control; RT‑qPCR, reverse transcription‑quantitative PCR; NFAT5, nuclear factor of activated T cells 5.

Article Snippet: containing the coding region for myc‐tagged nFaT5 (eGFP removed during construction; cat. no. 13627; addgene, inc.) was a kind gift from dr anjana rao (department of Pathology, Harvard Medical School) (15). peGFP‐n1, the empty vector, was a kind gift from dr Ye Qiu (college of Biology, Hunan university). pSi‐check2‐hrluc‐nF‐κB‐firefly, the nF‐κB luciferase reporter construct, was a gift from dr Qing deng (department of Biological Sciences, Purdue university) (cat. no. 106979; addgene, inc.) (16).

Techniques: Cell Culture, Incubation, Microscopy, Viability Assay, Western Blot, Control

Journal: Molecular medicine reports

Article Title: Elevation of hypertonicity‑induced protein NFAT5 promotes apoptosis of human umbilical vein endothelial cells through the NF‑κB pathway.

doi: 10.3892/mmr.2021.11823

Figure Lengend Snippet: Figure 2. Overexpression of NFAT5 induces caspase‑3 cleavage in HUVECs and causes cell death. HUVECs were transfected with pEGFP‑NFAT5 (NFAT5) or pEGFP‑N1 empty vector (Vector). At 48 h post transfection, the cells were subjected to (A) western blotting detection of NFAT5 and caspase‑3 cleavage, (B) phase‑contrast microscopy (scale bar, 200 µm) and (C) MTS cell viability assay. β‑actin was used as a loading control. Cell viability is presented as a percentage of the Ctr. n=9 in each group. **P<0.01, Student's t‑test. NFAT5, nuclear factor of activated T cells 5; HUVEC, human umbilical vein endothelial cell; Iso, isotonic medium; Hyp, hypertonic medium.

Article Snippet: containing the coding region for myc‐tagged nFaT5 (eGFP removed during construction; cat. no. 13627; addgene, inc.) was a kind gift from dr anjana rao (department of Pathology, Harvard Medical School) (15). peGFP‐n1, the empty vector, was a kind gift from dr Ye Qiu (college of Biology, Hunan university). pSi‐check2‐hrluc‐nF‐κB‐firefly, the nF‐κB luciferase reporter construct, was a gift from dr Qing deng (department of Biological Sciences, Purdue university) (cat. no. 106979; addgene, inc.) (16).

Techniques: Over Expression, Transfection, Plasmid Preparation, Western Blot, Microscopy, Viability Assay, Control

Journal: Molecular medicine reports

Article Title: Elevation of hypertonicity‑induced protein NFAT5 promotes apoptosis of human umbilical vein endothelial cells through the NF‑κB pathway.

doi: 10.3892/mmr.2021.11823

Figure Lengend Snippet: Figure 3. Knockdown of NFAT5 inhibits the apoptosis of HUVECs. HUVECs were transfected with siNFAT5 or scrambled control siRNA (Control). At 40 h post‑transfection, the culture medium was replaced with hypertonic medium or isotonic medium as a control. After another 8 h of incubation, the cells were subjected to (A) western blotting detection of NFAT5 and caspase‑3 cleavage and (B) MTS cell viability assay. β‑actin was used as a loading control. Cell viability is presented as a percentage of the Ctr. n=9 in each group. *P<0.05, **P<0.01, Student's t‑test. NFAT5, nuclear factor of activated T cells 5; HUVEC, human umbilical vein endothelial cell; si, small interfering; Iso, isotonic medium; Hyp, hypertonic medium.

Article Snippet: containing the coding region for myc‐tagged nFaT5 (eGFP removed during construction; cat. no. 13627; addgene, inc.) was a kind gift from dr anjana rao (department of Pathology, Harvard Medical School) (15). peGFP‐n1, the empty vector, was a kind gift from dr Ye Qiu (college of Biology, Hunan university). pSi‐check2‐hrluc‐nF‐κB‐firefly, the nF‐κB luciferase reporter construct, was a gift from dr Qing deng (department of Biological Sciences, Purdue university) (cat. no. 106979; addgene, inc.) (16).

Techniques: Knockdown, Transfection, Control, Incubation, Western Blot, Viability Assay

Journal: Molecular medicine reports

Article Title: Elevation of hypertonicity‑induced protein NFAT5 promotes apoptosis of human umbilical vein endothelial cells through the NF‑κB pathway.

doi: 10.3892/mmr.2021.11823

Figure Lengend Snippet: Figure 4. Overexpression of NFAT5 or hyptertonic condition enhances the activity of NF‑κB in HUVECs. (A) HUVECs were cotransfected with pSI‑Check2‑hRluc‑NF‑κB‑firefly and pEGFP‑NFAT5 (NFAT5) or pEGFP‑N1 empty vector (Vector). At 48 h post transfection, the cells were lysed and subjected to dual‑luciferase assay. (B) HUVECs were co‑transfected with pSI‑Check2‑hRluc‑NFκB‑firefly and scrambled siRNA or NFAT5 siRNA. At 40 h post transfection, the culture medium was replaced with hypertonic medium or isotonic medium as a control. After another 4 h of incubation, the cells were lysed and subjected to dual‑luciferase assay. The luminescence of firefly luciferase was normalized by the luminescence of Renilla luciferase to generate RLU. (C) HUVECs were transfected with pEGFP‑NFAT5 (NFAT5) or pEGFP‑N1 empty vector. At 48 h post transfection, the cells were lysed and subjected to RT‑qPCR detection of VCAM‑1, ICAM‑1 and iNOS. GAPDH mRNA levels were used as the endogenous control to normalize the data. All RT‑qPCR experiments were performed in triplicate and the results were presented as the fold change of each vector group. n=9 in each group. *P<0.05, **P<0.01, Student's t‑test. (D) HUVECs were transfected with pEGFP‑NFAT5 (NFAT5) or pEGFP‑N1 empty vector. At 48 h post transfection, the cells were subjected to western blotting detection of NFAT5 and Bcl‑2. β‑actin was used as a loading control. NFAT5, nuclear factor of activated T cells 5; HUVEC, human umbilical vein endothelial cell; si, small interfering; RLU, relative luminescence unit; RT‑qPCR, reverse transcription‑quantitative PCR; VCAM‑1, vascular cell adhesion molecule 1; ICAM‑1, intercellular adhesion molecule 1; iNOS, inducible nitric oxide synthase; Vector, empty vector.

Article Snippet: containing the coding region for myc‐tagged nFaT5 (eGFP removed during construction; cat. no. 13627; addgene, inc.) was a kind gift from dr anjana rao (department of Pathology, Harvard Medical School) (15). peGFP‐n1, the empty vector, was a kind gift from dr Ye Qiu (college of Biology, Hunan university). pSi‐check2‐hrluc‐nF‐κB‐firefly, the nF‐κB luciferase reporter construct, was a gift from dr Qing deng (department of Biological Sciences, Purdue university) (cat. no. 106979; addgene, inc.) (16).

Techniques: Over Expression, Activity Assay, Plasmid Preparation, Transfection, Control, Incubation, Luciferase, Western Blot

Journal: PLoS Pathogens

Article Title: Cleavage of osmosensitive transcriptional factor NFAT5 by Coxsackieviral protease 2A promotes viral replication

doi: 10.1371/journal.ppat.1006744

Figure Lengend Snippet: SV40 human cardiomyocytes and HeLa cells were infected with CVB3 at an MOI of 40 or 10, respectively, or sham-infected with PBS and harvested at the indicated time points pi. Cellular proteins and RNAs were extracted for Western blot analysis of NFAT5 protein (A, C) and qPCR measurement of NFAT5 mRNA (B, D) , respectively. In qPCR assay, the result was shown as the relative level of each mRNA normalized to the level of GAPDH mRNA in the sample. Three biological replicates were performed for each assay. (E) 4-week-old A/J mice were infected with CVB3 at 10 5 pfu (plaque forming unit) or sham-infected with PBS. At 6 days pi, the mice were sacrificed and the heart tissue was homogenized for Western blot analysis of NFAT5 protein. β-actin was used as a loading control. Quantitation of NFAT5 protein was conducted by densitometry analysis using the NIH ImageJ program (right panel). Three biological replicates were performed and the result was subjected to statistical analysis.

Article Snippet: The NFAT5 reporter plasmid pGL-TonE-luciferase and the control Renilla reporter plasmid pRL-PolIII were kind gifts from Dr. Anjana Rao (Addgene plasmid # 14110) [ ] and Dr. Norbert Perrimon (Addgene plasmid # 37380) [ ], respectively.

Techniques: Infection, Western Blot, Control, Quantitation Assay

Journal: PLoS Pathogens

Article Title: Cleavage of osmosensitive transcriptional factor NFAT5 by Coxsackieviral protease 2A promotes viral replication

doi: 10.1371/journal.ppat.1006744

Figure Lengend Snippet: HeLa cells were treated with 10 μM MG132 (A) or 25 μM z-VAD-fmk (B) and then infected with CVB3 at an MOI of 10. At indicated time points pi, the cellular proteins were subjected to Western blot analysis of NFAT5 and other proteins using the indicated antibodies. β-actin was used as a loading control. (C) HeLa cells were infected by CVB3 at an MOI of 10 for 4 and 6 h and then subjected to Western blot analysis using an antibody against the N-terminal epitope of NFAT5. (D) HeLa cells transfected with a plasmid expressing the 6*myc-NFAT5 fusion protein (upper panel) were infected with CVB3 or sham-infected as described above and subjected to Western blot analysis using an antibody against myc tag (lower panel). (E) HeLa cells expressing myc-NFAT5 were transfected with pIRES-2A (2A), pIRES-3C (3C) or vector only (V). At 36 h pt, the cells were subjected to Western blot analysis using an antibody against myc tag. The cells infected with CVB3 or sham-infected with PBS were used as controls. Arrows indicate the 3C cleavage bands. (F) Tissue homogenate from mouse heart was incubated with recombinant 2A or 3C for 8 h. Then the mouse NFAT5 (mNFAT5) N-terminal epitope was detected by Western blot using a specific antibody.

Article Snippet: The NFAT5 reporter plasmid pGL-TonE-luciferase and the control Renilla reporter plasmid pRL-PolIII were kind gifts from Dr. Anjana Rao (Addgene plasmid # 14110) [ ] and Dr. Norbert Perrimon (Addgene plasmid # 37380) [ ], respectively.

Techniques: Infection, Western Blot, Control, Transfection, Plasmid Preparation, Expressing, Incubation, Recombinant

Journal: PLoS Pathogens

Article Title: Cleavage of osmosensitive transcriptional factor NFAT5 by Coxsackieviral protease 2A promotes viral replication

doi: 10.1371/journal.ppat.1006744

Figure Lengend Snippet: (A) The potential 2A cleavage sites predicted by using the NetPicoRNA 1.0 program showing the cleavage position (Pos), cleavage score (Clv) and surface score (Surf). The arrow indicates the exact cleavage locus. (B) Alignment of the sequences around G503 of NFAT5 from different species. (C) HeLa cells were transfected with a plasmid expressing myc-NFAT5, myc-NFAT5 G503A or myc-NFAT5 G650A and then infected with CVB3 at an MOI of 10 for 6 h. Then the cell lysates were subjected to Western blot analysis using an anti-myc antibody. Arrows indicate the 3C cleavage bands. (D) HeLa cells were co-transfected with pIRES-2A and a plasmid expressing WT or mutant NFAT5 G503A . At 36 h pt, the cell lysates were subjected to Western blot analysis using an anti-myc antibody.

Article Snippet: The NFAT5 reporter plasmid pGL-TonE-luciferase and the control Renilla reporter plasmid pRL-PolIII were kind gifts from Dr. Anjana Rao (Addgene plasmid # 14110) [ ] and Dr. Norbert Perrimon (Addgene plasmid # 37380) [ ], respectively.

Techniques: Transfection, Plasmid Preparation, Expressing, Infection, Western Blot, Mutagenesis

Journal: PLoS Pathogens

Article Title: Cleavage of osmosensitive transcriptional factor NFAT5 by Coxsackieviral protease 2A promotes viral replication

doi: 10.1371/journal.ppat.1006744

Figure Lengend Snippet: (A) HeLa cells were transfected with NFAT5 siRNAs or scrambled control siRNAs (Ctr siRNA) and then infected with CVB3. At 4 and 6 h pi, the cell lysates were subjected to Western blot analysis of NFAT5 and VP1. Quantitation of VP1 was conducted by densitometry scan using the NIH ImageJ program and normalized against β-actin control (right panel). (B) HeLa cells were transfected with plasmids expressing WT myc-NFAT5 or myc-NFAT5 G503A , infected with CVB3 and then harvested for VP1 detection and quantification as described above (right panel). (C) HeLa cells were transfected with plasmids expressing p70-NFAT5 tagged with FLAG and then infected with CVB3 for VP1 detection and quantification (right panel) at 6 h pi. (D-E ) HeLa cells were transfected with plasmids expressing FL-NFAT5, NFAT5 G503A (NFAT5-G503A) or p70-NFAT5 and then infected with CVB3. At 6 h pi, the cellular RNAs were extracted for qPCR measurement of CVB3 genomic RNA level (normalized to GAPDH as described in ) (D) and the medium was collected for plaque assay to measure viral titre (E ). Three biological replicates were performed for each assay and the result was subjected to statistical analysis.

Article Snippet: The NFAT5 reporter plasmid pGL-TonE-luciferase and the control Renilla reporter plasmid pRL-PolIII were kind gifts from Dr. Anjana Rao (Addgene plasmid # 14110) [ ] and Dr. Norbert Perrimon (Addgene plasmid # 37380) [ ], respectively.

Techniques: Transfection, Control, Infection, Western Blot, Quantitation Assay, Expressing, Plaque Assay

Journal: PLoS Pathogens

Article Title: Cleavage of osmosensitive transcriptional factor NFAT5 by Coxsackieviral protease 2A promotes viral replication

doi: 10.1371/journal.ppat.1006744

Figure Lengend Snippet: (A) HeLa cells were co-transfected with pEGFP-myc-NFAT5 and pcDNA3.1(-)-p70-NFAT5 or pcDNA3.1(-) empty vector. Then the cells were infected with CVB3. At 4 and 6 h pi, the cells were fixed and immunostained using a specific antibody against the myc tag and observed by confocal microscopy. (B) HeLa cells were co-transfected with the luciferase reporter constructs pGL-TonE-luciferase or pRL-polIII and the plasmids expressing FL NFAT5, p70-NFAT5, NFAT5 DBD or pEGFP empty vector. At 48 h pt, the cell lysates were subjected to dual luciferase assay detecting the activity of the firefly luciferase (Fluc) and the renilla luciferase (Rluc). The relative activity (FLu/RLu) was determined after normalization against the vector control. (C, D) HeLa cells were transfected with plasmids expressing FL NFAT5 or p70-NFAT5 and then infected with CVB3. At 6 h pi, the cellular RNAs were extracted for qPCR measurement of mRNA level of TauT (C) and SMIT (D) (normalized to GAPDH as described in ). Vector-transfected cells were used as a control. Three biological replicates were performed for each assay and the results was subjected to statistical analysis.

Article Snippet: The NFAT5 reporter plasmid pGL-TonE-luciferase and the control Renilla reporter plasmid pRL-PolIII were kind gifts from Dr. Anjana Rao (Addgene plasmid # 14110) [ ] and Dr. Norbert Perrimon (Addgene plasmid # 37380) [ ], respectively.

Techniques: Transfection, Plasmid Preparation, Infection, Confocal Microscopy, Luciferase, Construct, Expressing, Activity Assay, Control

Journal: PLoS Pathogens

Article Title: Cleavage of osmosensitive transcriptional factor NFAT5 by Coxsackieviral protease 2A promotes viral replication

doi: 10.1371/journal.ppat.1006744

Figure Lengend Snippet: HeLa cells were transfected with plasmids expressing FL NFAT5 or p70-NFAT5 and then infected with CVB3. At 6 h pi, the cellular RNAs were extracted for qPCR measurement of mRNA levels of Hsp70-2 (A) , TNFα (B) and iNOS (C) (normalized to GAPDH as described in ). (D) HeLa cells transfected with pEGFP empty vector (V) or pEGFP-myc-NFAT5 were treated with 4 mM 1400W or DMSO control and then infected with CVB3 for VP1 detection and quantification (lower panel) as described in . Three biological replicates were performed for each assay and the result was subjected to statistical analysis.

Article Snippet: The NFAT5 reporter plasmid pGL-TonE-luciferase and the control Renilla reporter plasmid pRL-PolIII were kind gifts from Dr. Anjana Rao (Addgene plasmid # 14110) [ ] and Dr. Norbert Perrimon (Addgene plasmid # 37380) [ ], respectively.

Techniques: Transfection, Expressing, Infection, Plasmid Preparation, Control

Journal: PLoS Pathogens

Article Title: Cleavage of osmosensitive transcriptional factor NFAT5 by Coxsackieviral protease 2A promotes viral replication

doi: 10.1371/journal.ppat.1006744

Figure Lengend Snippet: ( A) HCMs were transfected with NFAT5 siRNA or control (Ctr) siRNA. At 36 h pt, the cells were treated with 200 mM mannitol solution or PBS and then infected with CVB3 at an MOI of 40 or sham-infected with PBS. At 10 h pi, cells were subjected to Western blot analysis of NFAT5, iNOS and VP1. ( B ) HCMs were cotransfected with plasmids expressing myc-NFAT5 or empty vector pEGFP and iNOS siRNA or Ctr siRNA. At 36 h pt, the cells were infected with CVB3 at an MOI of 40 or sham-infected with PBS. At 10 h pi, cells were subjected to Western blot analysis of myc-NFAT5, iNOS and VP1. Quantification of iNOS and VP1 was performed as described in (lower panels). Three biological replicates were conducted for each assay and subjected to statistical analysis.

Article Snippet: The NFAT5 reporter plasmid pGL-TonE-luciferase and the control Renilla reporter plasmid pRL-PolIII were kind gifts from Dr. Anjana Rao (Addgene plasmid # 14110) [ ] and Dr. Norbert Perrimon (Addgene plasmid # 37380) [ ], respectively.

Techniques: Transfection, Control, Infection, Western Blot, Expressing, Plasmid Preparation

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: European journal of immunology

Article Title: The xanthine oxidase-NFAT5 pathway regulates macrophage activation and TLR-induced inflammatory arthritis.

doi: 10.1002/eji.201343669

Figure Lengend Snippet: Figure 1. NFAT5 differentially regulates NO, IL-6, and IL-10 production by macrophages. (A) RAW 264.7 cells were stably transfected with lentiviral particles harboring NFAT5 shRNA or scrambled shRNA. The NFAT5 reporter gene was then transiently co-transfected into the cells, which were then stimulated with heat-inactivated E. coli (3 × 106 CFU/mL). GFP expression was assessed by fluorescence microscopy (left). Reduction in NFAT5 expression was confirmed by Western blot analysis (right), using α-tubulin as a loading control. (B) After stable knockdown with NFAT5 shRNA, RAW 264.7 macrophages were stimulated with LPS (1 μg/mL; left panel and 5 μg/mL; right panel), E. coli or peptidoglycan (PG; 10 μg/mL) for 24 h, and nitrite levels in the culture supernatants were measured by the Griess reaction. The degree of cell viability was assessed using an MTT assay. Data are presented as nitrite or IL-6 concentration relative to the percentage of the viability of NFAT5 shRNA-treated cells to the viability of control cells. p < 0.01 versus control (scrambled) shRNA. (C) RAW 264.7 macrophages were transfected stably with NFAT5 shRNA, and then stimulated with LPS (0.1, 1, 5, and 10 μg/mL) or PG (0.1, 1, 5, and 10 μg/mL) for 24 h. IL-6 production was determined by ELISA. *p < 0.01 versus control shRNA-transfected cells. (D) Macrophages were stably transfected with an NFAT5 decoy or NDBD, and then stimulated with the indicated concentrations of LPS for 24 h. IL-6 production in response to an NFAT5 decoy (left panel) encompassing the NFAT5 consensus sequence or NDBD of NFAT5 (right panel), was assessed by ELISA. NDBD fused to the nuclear localization sequence (NLS) sized 27 kDa was identified by Western blot analysis using anti-NFAT5 antibodies (right upper corner of the graph). β-actin was used as a loading control (data not shown). *p < 0.01 versus control NFAT5 decoy (left) or mock DNA (right). (E) IL-10 production by RAW 264.7 cells was assessed 24 h after stimulation with LPS or PG. p < 0.01 versus control shRNA. (F) RAW 264.7 macrophages were stimulated with LPS (5 μg/mL) or PG (10 μg/mL) for 24 h, and then COX-2 and iNOS expression in the cells was determined by Western blot analysis. α-tubulin was used as a loading control. (B–D) Data are shown as mean + SD and are representative of one of at least three independent experiments, each performed in triplicate. (F) Data are representative of one of three independent experiments with similar results. Comparison of the data between groups was performed by the Mann–Whitney U-test.

Article Snippet: To make NFAT5-deficient macrophages, RAW 264.7 macrophages were seeded to 20% confluence in 12-well plates and then transfected with NFAT5 shRNA harboring lenti-viral particle (Santa Cruz Biotechnology) in the presence of polybrene (5 μg/mL).

Techniques: Stable Transfection, Transfection, shRNA, Expressing, Microscopy, Western Blot, Control, Knockdown, MTT Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Sequencing, Comparison, MANN-WHITNEY

Journal: European journal of immunology

Article Title: The xanthine oxidase-NFAT5 pathway regulates macrophage activation and TLR-induced inflammatory arthritis.

doi: 10.1002/eji.201343669

Figure Lengend Snippet: Figure 2. p38 MAPK is required for TLR-triggered NFAT5 activity. (A) RAW 264.7 macrophages stably transfected with the NFAT5 reporter gene were stimulated with LPS (5 μg/mL) or heat-inactivated E. coli (3 × 106 CFU/mL) for 24 h in the presence of SB203580 (30 μM), PD98059 (30 μM), or SP600125 (20 μM). NFAT5-dependent reporter activity was then determined by flow cytometry. Based on forward-scatter (FSC) and side-scatter (SSC) characteristics, the live gated cells were analyzed by flow cytometry, and NFAT5-dependent GFP levels were assessed by measuring mean fluorescence intensity (MFI). Data in the bottom panel depict the mean + SD of three independent experiments in duplicate. *p < 0.01 versus media, LPS or E. coli alone. (B) RAW 264.7 macrophages were stimulated with LPS (1 μg/mL) for 24 h in the absence (Con) or presence of SB203580 (1–25 μM). NFAT5 protein expression was determined by Western blot analysis. β-actin was used as a loading control. Data are representative of three independent experiments. (C) RAW 264.7 cells were stimulated with LPS (1 μg/mL) for 12 h in the presence of SB203580 (5 μM). NFAT5 mRNA expression was determined by real-time PCR analysis. Data are presented as the mean + SD of three independent experiments in duplicate. *p < 0.05 versus LPS alone by the Mann–Whitney U-test. (D) RAW 264.7 macrophages were stimulated with LPS (5 μg/mL) in the absence or presence of SB203580 (30 μM) for 24 h. Fractionated cell lysates were then subjected to Western blot analysis using α-tubulin and NMP p84 as a loading control; C: cytoplasmic fraction, N: nuclear fraction. Data are representative of three independent experiments. (E) NFAT5 reporter gene and DN p38α gene were transiently co-transfected into macrophages, and the GFP activity for NFAT5 was determined by flow cytometry 24 h after stimulation with LPS (5 μg/mL). Based on FSC and SSC characteristics, the live cells were gated. Data are the mean + SD of three independent experiments in duplicate. *p < 0.01 versus media or LPS alone by the Mann–Whitney U-test. (F) NFAT5 protein expression was determined by Western blot analysis in RAW 264.7 macrophages transfected with DN p38α genes 24 h after stimulation with LPS (1 μg/mL). β-actin was used as a loading control. Data are representative of three independent experiments.

Article Snippet: To make NFAT5-deficient macrophages, RAW 264.7 macrophages were seeded to 20% confluence in 12-well plates and then transfected with NFAT5 shRNA harboring lenti-viral particle (Santa Cruz Biotechnology) in the presence of polybrene (5 μg/mL).

Techniques: Activity Assay, Stable Transfection, Transfection, Cytometry, Expressing, Western Blot, Control, Real-time Polymerase Chain Reaction, MANN-WHITNEY

Journal: European journal of immunology

Article Title: The xanthine oxidase-NFAT5 pathway regulates macrophage activation and TLR-induced inflammatory arthritis.

doi: 10.1002/eji.201343669

Figure Lengend Snippet: Figure 4. Activation of the XO–p38–NFAT5 axis in primary macrophages upon TLR ligation. (A) NFAT5 expression was determined by Western blot analysis in (i) mouse (C57BL/6) peritoneal macrophages or (ii) human macrophages differentiated from peripheral blood monocytes of healthy subjects, both of which were stimulated with LPS (0.01 and 1 μg/mL) or peptidoglycan (PG; 0.01 and 1 μg/mL) for 24 h. β-actin was used as a loading control. Data are representative of three independent experiments. (B) Peritoneal macrophages of C57BL/6 mice were obtained 3 days after intraperitoneal injection of 3% thioglycollate. The cells were transiently transfected with the NFAT5 reporter gene for 24 h, and then stimulated with LPS (5 μg/mL), PG (10 μg/mL), or E. coli (3 × 106 CFU/mL) for 24 h. NFAT5-dependent reporter activity was determined by flow cytometry. The live cells were gated on the basis of FSC and SSC properties. Data in the bottom panel show the mean + SD of three independent experiments in duplicate. (C) Expression of IL-6, nitrite, and TNF-α in peritoneal macrophages of NFAT5+/+ and NFAT5+/−mice (n = 7 each, from three independent experiments) injected with 3% thioglycollate. In the top panel, IL-6 and TNF-α expressions in the live gated cells were determined by flow cytometry analysis, which was performed immediately after isolation of peritoneal cells of NFAT5+/+ and NFAT5+/−mice. In the bottom panel, nitrite and IL-6 levels in the culture supernatants were determined by ELISA 24 h after stimulation of sorted peritoneal macrophages with LPS (0.01 μg/mL). ELISA data are shown as mean + SD. *p < 0.05 versus NFAT5+/+ mice. (D) Peritoneal macrophages of C57BL/6 mice (n = 4, from two independent experiments) were stimulated with LPS (0.01 μg/mL) in the absence or presence of allopurinol (Allo; 1 mM). Nitrite and IL-6 concentrations in the culture supernatants were determined by ELISA. Data are shown as mean + SD. *p < 0.05 versus LPS alone. (E) Human macrophages differentiated from peripheral monocytes of healthy volunteers were stimulated with LPS (1 μg/mL) for 24 h in the presence of SB203580 (5 and 10 μM), allopurinol (Allo; 5 mM), or NAC (10 and 20 mM). NFAT5 expression was determined by Western blot analysis. β-actin was used as a loading control. Con: control cells treated with neither LPS nor SB203580. Data are the representative of three independent experiments. (F) Normal human macrophages (n = 6) and synovial macrophages of RA patients (n = 4) were stimulated with LPS (1 μg/mL) for 12 h after transfection with a lentivirus expressing NFAT5 shRNA or control shRNA (Con shRNA) for 48 h. NFAT5 mRNA and protein expressions were determined by real-time PCR and Western blot analysis, respectively (left panel, lane 1: control shRNA, lane 2: NFAT5 shRNA). IL-6 production in the culture supernatants was assessed by ELISA (right panel). Data are shown as mean + SD. **p < 0.001, ***p < 0.0001 versus Con shRNA. (G) Normal macrophages (n = 6) and macrophages of RA patients (n = 8) isolated from synovial fluids were stimulated with LPS (1 μg/mL) for 24 h in the absence or presence of Allo (1 and 5 mM). Levels of IL-6 in the culture supernatants were determined by ELISA and data are shown as mean + SD. *p < 0.05 versus vehicle treatment. (C, D, F, and G) Statistical analysis of the data was performed by the Mann–Whitney U-test.

Article Snippet: To make NFAT5-deficient macrophages, RAW 264.7 macrophages were seeded to 20% confluence in 12-well plates and then transfected with NFAT5 shRNA harboring lenti-viral particle (Santa Cruz Biotechnology) in the presence of polybrene (5 μg/mL).

Techniques: Activation Assay, Ligation, Expressing, Western Blot, Control, Injection, Transfection, Activity Assay, Cytometry, Isolation, Enzyme-linked Immunosorbent Assay, shRNA, Real-time Polymerase Chain Reaction, MANN-WHITNEY

Journal: European journal of immunology

Article Title: The xanthine oxidase-NFAT5 pathway regulates macrophage activation and TLR-induced inflammatory arthritis.

doi: 10.1002/eji.201343669

Figure Lengend Snippet: Figure 6. In vivo evidence for XO regulation of NFAT5 activity and IL-6 production. (A) Protocol: In the top portion, Matrigel containing RAW 264.7 cells transfected stably with NFAT5-RFP reporter were mixed with LPS (10 μg/mL), and subcutaneously injected into the back skin of BALB/c mice. RFP fluorescence was captured by an in vivo imaging system 16 h after implantation. In the bottom portion, Matrigels with NFAT5-RFP reporter in the absence of LPS were subcutaneously injected into BALB/c mice. After new blood vessels grew inside the Matrigel on day 9, LPS (10 mg/kg) was injected intraperitoneally into the mice. Matrigel was removed 16 h after LPS injection, and the RFP activity was then determined by in vivo imaging. (B) LPS was premixed (in the top panel) with Matrigel or injected systemically into BALB/c mice (in the middle and bottom panels) and the intensity of fluorescence for each Matrigel complex was measured by a Maestro 2 imaging system. Data are representative of two independent experiments. (C) A premix of allopurinol (1 mM) with LPS-treated Matrigel was implanted into BALB/c mice. RFP fluorescence was captured by an in vivo imaging system 16 h after implantation. (D) On day 9, LPS (10 mg/kg) plus allopurinol (25 mg/kg) was intraperitoneally injected into BALB/c mice. The RFP intensity for each Matrigel complex was measured by in vivo imaging. Data are representative of two independent experiments. (E) IL-6 concentrations in the sera of the same mice described in (D) were measured by ELISA. Data are shown as mean + SD of independent two experiments (n = 6 for each group). *p < 0.01 versus LPS alone by the Mann–Whitney U-test.

Article Snippet: To make NFAT5-deficient macrophages, RAW 264.7 macrophages were seeded to 20% confluence in 12-well plates and then transfected with NFAT5 shRNA harboring lenti-viral particle (Santa Cruz Biotechnology) in the presence of polybrene (5 μg/mL).

Techniques: In Vivo, Activity Assay, Transfection, Stable Transfection, Injection, In Vivo Imaging, Imaging, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Frontiers in Physiology

Article Title: NFAT5 Isoform C Controls Biomechanical Stress Responses of Vascular Smooth Muscle Cells

doi: 10.3389/fphys.2018.01190

Figure Lengend Snippet: Analysis of NFAT5 mRNA expression in VSMCs. Localization of NFAT5 protein was detected by immunofluorescence ( A , scale bar: 100 μm) and NFAT5 mRNA expression was analyzed by semi-quantitative PCR ( B , ∗ p < 0.05, n = 3, unpaired, 2-tailed Student’s t -test; the expression of the housekeeping gene RPL32 served as internal standard) in resting and stretch-stimulated (24 h) cultured HUASMCs. After treatment (24 h) with actinomycin-D NFAT5 mRNA expression declined ( C , ActD; ∗∗∗ p < 0.001, ∗∗ p < 0.01, n = 4; one-way ANOVA and Tukey post-test).

Article Snippet: Plasmids (pCMV6) encoding for Myc-DDK-tagged NFAT5 were bought from Origene (NFAT5_isoform A: RC219340; NFAT5-isoform C: RC216142).

Techniques: Expressing, Immunofluorescence, Real-time Polymerase Chain Reaction, Cell Culture

Journal: Frontiers in Physiology

Article Title: NFAT5 Isoform C Controls Biomechanical Stress Responses of Vascular Smooth Muscle Cells

doi: 10.3389/fphys.2018.01190

Figure Lengend Snippet: Analysis of NFAT5 protein abundance upon inhibition of the proteasome. HUASMCs were treated (24 h) with increasing concentrations of bortezomib and NFAT5 was detected by immunostaining. The percentage of NFAT5-positive nuclei were quantified by automated image analysis ( A , ∗∗∗ p < 0.001, n = 3, one-way ANOVA and Tukey post-test; scale bar 100 μm). NFAT5 protein abundance was determined after treatment with 10 nM bortezomib (24 h) in nuclear (B) and cytosolic ( C ) fractions of cell lysates ( B,C , ∗∗∗ p < 0.001/ ∗∗ p < 0.01 vs. DMSO, n = 3, unpaired, 2-tailed Student’s t -test; histone H3 and tubulin served as loading controls).

Article Snippet: Plasmids (pCMV6) encoding for Myc-DDK-tagged NFAT5 were bought from Origene (NFAT5_isoform A: RC219340; NFAT5-isoform C: RC216142).

Techniques: Inhibition, Immunostaining

Journal: Frontiers in Physiology

Article Title: NFAT5 Isoform C Controls Biomechanical Stress Responses of Vascular Smooth Muscle Cells

doi: 10.3389/fphys.2018.01190

Figure Lengend Snippet: Biomechanical stretch induces NFAT5c translocation into the nucleus. Expression of NFAT5 isoforms after stretch was quantitated by semi-quantitative PCR ( A , ∗ p < 0.05 vs. control, n = 3, unpaired, 2-tailed Student’s t -test, n.s.: not significant). DDK-specific immunostaining was used for detection of NFAT5 isoform localization in HUASMCs exposed to biomechanical stretch for 24 h, white arrows indicate localization of the nuclei, scale bar: 50 μm). Automated quantification of DDK-positive nuclei in transfected cells 24 h after stretch ( B , ∗∗∗ p < 0.001, ∗ p < 0.05, n = 3, one-way ANOVA and Tukey post-test).

Article Snippet: Plasmids (pCMV6) encoding for Myc-DDK-tagged NFAT5 were bought from Origene (NFAT5_isoform A: RC219340; NFAT5-isoform C: RC216142).

Techniques: Translocation Assay, Expressing, Real-time Polymerase Chain Reaction, Immunostaining, Transfection

Journal: Frontiers in Physiology

Article Title: NFAT5 Isoform C Controls Biomechanical Stress Responses of Vascular Smooth Muscle Cells

doi: 10.3389/fphys.2018.01190

Figure Lengend Snippet: Analysis of nuclear translocation of NFAT5 upon knockdown of ABL1. HUASMCs were transfected with control (scrambled) siRNA or ABL1-specific siRNA (ABL1 Silencer ® Select Validated siRNA, S864, Ambion, Applied Biosystem, Thermo Fisher Scientific, Darmstadt, Germany) which decreased ABL1 expression by ∼50% as evidenced by PCR analyses (A) . Transfected HUASMCs were exposed to biomechanical stretch for 24 h. NFAT5-specific immunofluorescence was detected by automated image analysis to assess the percentage of NFAT5-positive nuclei ( B , ∗ p < 0.05, one-way ANOVA and Tukey post-test; control siRNA n = 5, ABL1 siRNA n = 10). Dasatinib (inhibitor of BCR-ABL kinases, 5 nM) treatment of stretch-stimulated HUASMCs decreased the number of NFAT5 positive nuclei as evidenced by automated immunofluorescence analyses ( C , ∗ p < 0.05, one-way ANOVA and Tukey post-test, n = 3). Additionally, HUASMCs were treated with Dasatinib (Dasa; 5 nM) or DMSO for 1 h and exposed to biomechanical stretch for 24 h. Accumulation of NFAT5 in the nucleus was determined in nuclear lysates by immunoblot techniques with (nuclear) histone H3 as loading marker ( D , ∗∗∗ p < 0.05, n.s.: not significant, one-way ANOVA and Tukey post-test, n = 3).

Article Snippet: Plasmids (pCMV6) encoding for Myc-DDK-tagged NFAT5 were bought from Origene (NFAT5_isoform A: RC219340; NFAT5-isoform C: RC216142).

Techniques: Translocation Assay, Transfection, Expressing, Immunofluorescence, Western Blot, Marker

Journal: Frontiers in Physiology

Article Title: NFAT5 Isoform C Controls Biomechanical Stress Responses of Vascular Smooth Muscle Cells

doi: 10.3389/fphys.2018.01190

Figure Lengend Snippet: Overexpression of NFAT5c promotes its nuclear accumulation and activates VSMCs. HUASMCs were transduced with adenovirus to overexpress NFAT5c (Ad-N5) or (Ad-PL) control plasmid, N FAT5 -expression was quantitated by qRT-PCR, immunostaining and immunoblot of nuclear and cytosolic lysates 48 h after transfection ( A–C , ∗∗ p < 0.01 vs. Ad-PL, n = 6, unpaired, 2-tailed Student’s t -test; histone H3 and β-actin served as loading controls). Expression levels of NFAT5 target gene TNC was assessed by qRT-PCR ( D , ∗∗ p < 0.01 vs. Ad-PL, n = 6, unpaired, 2-tailed Student’s t -test). The migratory capacity of collagen-embedded NFAT5c -overexpressing spheroids was analyzed by assessing the number of sprouts, mean sprout length and cumulative sprout length after 24 h ( E , ∗∗∗ p < 0.001 vs. Ad-PL, n = 10 spheroids/condition, 1 out of 3 independent experiments with comparable results, unpaired, 2-tailed Student’s t -test; scale bar: 200 μm). Proliferation of NFAT5c-overexpressing HUASMCs 72 h after transfection by immunostaining of the proliferation marker Ki67 ( F , ∗ p < 0.05, n.s.: not significant, vs. Ad-PL, n = 4, unpaired, 2-tailed Student’s t -test, scale bar: 100 μm).

Article Snippet: Plasmids (pCMV6) encoding for Myc-DDK-tagged NFAT5 were bought from Origene (NFAT5_isoform A: RC219340; NFAT5-isoform C: RC216142).

Techniques: Over Expression, Transduction, Plasmid Preparation, Expressing, Quantitative RT-PCR, Immunostaining, Western Blot, Transfection, Marker

Journal: Frontiers in Physiology

Article Title: NFAT5 Isoform C Controls Biomechanical Stress Responses of Vascular Smooth Muscle Cells

doi: 10.3389/fphys.2018.01190

Figure Lengend Snippet: Gene set enrichment analyses (GSEA) based on the KEGG pathway database. HUASMCs from three donors were treated with NFAT5-specific or control siRNA and exposed to biomechanical stretch for 24 h. RNA was isolated and processed for genome array analysis (HuGene-1 0-st-v1 array, Affymetrix). The table (A) lists all significantly DOWN-regulated gene sets associated with the indicated pathways/functions (log2-fold regulation, p < 0.05 NFAT5 knockdown vs. control, n = 3, one-way ANOVA; access# for full microarray data via Gene Expression Omnibus (GEO): GSE106274). Exemplary results of the subsequent gene set enrichment analyses (GSEA) for selected gene sets (enrichment plots) and the corresponding statistical values are shown [ B–D ; NES: normalized enrichment score; p: p -value (ANOVA); FDR: false discovery rate]. Additionally, the heat map corresponding to the enrichment plot for cell cycle-associated genes is shown.

Article Snippet: Plasmids (pCMV6) encoding for Myc-DDK-tagged NFAT5 were bought from Origene (NFAT5_isoform A: RC219340; NFAT5-isoform C: RC216142).

Techniques: Isolation, Microarray, Expressing

Journal: Frontiers in Physiology

Article Title: NFAT5 Isoform C Controls Biomechanical Stress Responses of Vascular Smooth Muscle Cells

doi: 10.3389/fphys.2018.01190

Figure Lengend Snippet: Knockdown of NFAT5 inhibits stretch-induced proliferation of HUASMCs. HUASMCs were treated with NFAT5-specific or control siRNA and exposed to biomechanical stretch for 24 h. Proliferation was assessed by detecting the proliferation marker Ki67 via immunofluorescence techniques (arrow) and determining the percentage of Ki67-positive nuclei ( ∗∗ p < 0.01, one-way ANOVA and Tukey post-test, n = 3; scale bar: 50 μm).

Article Snippet: Plasmids (pCMV6) encoding for Myc-DDK-tagged NFAT5 were bought from Origene (NFAT5_isoform A: RC219340; NFAT5-isoform C: RC216142).

Techniques: Marker, Immunofluorescence