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: Cell communication and signaling : CCS

Article Title: NFAT5 mediates hypertonic stress-induced atherosclerosis via activating NLRP3 inflammasome in endothelium.

doi: 10.1186/s12964-019-0406-7

Figure Lengend Snippet: Fig. 4 High-salt intake enhances NFAT5 expression and nuclear translocation in ECs. a Quantification of mRNA levels of NFAT5 in TA and AA of ApoE−/−mice fed with a normal or high-salt diet. b En face immunofluorescent staining of NFAT5 in ECs of TA and AA of ApoE−/−mice fed with a normal or high-salt diet. Circles mark the cells with nuclear NFAT5 expression. c, d mRNA and protein levels of NFAT5 in HUVECs exposed to iso- and hyper-osmotic media, evaluated by RT-qPCR and western blotting with β-actin as the internal control. All data were presented as mean ± SEM, N ≥3. *p < 0.05

Article Snippet: Recombinant adenovirus that containing the full cDNA sequence of NFAT5 (Ad-NFAT5), were purchased from Cyagen (Guangzhou, China).

Techniques: Expressing, Translocation Assay, Staining, Quantitative RT-PCR, Western Blot, Control

Journal: Cell communication and signaling : CCS

Article Title: NFAT5 mediates hypertonic stress-induced atherosclerosis via activating NLRP3 inflammasome in endothelium.

doi: 10.1186/s12964-019-0406-7

Figure Lengend Snippet: Fig. 5 High-salt activates NRLP3 inflammasome in ECs via NFAT5. a Immunoblot of NFAT5, caspase-1 p20, pro-IL-1β, and IL-1β p17, and quantification of caspase-1 activity and mature IL-1β in ECs treated with Adenovirus-null (Ad-null, 5 MOI) and Adenovirus-NFAT5 (Ad-NFAT5, 2 MOI or 5 MOI). See Additional file 1: Figure S4 for caspase-1 activity. b Immunoblot images of NFAT5, caspase-1 p20, pro-IL-1β, and IL-1β p17, and quantification of active caspase-1 and mature IL-1β in ECs treated with high-salt and transfected with Ctr siRNA or NFAT5 siRNA. c Immunoblot images of NLRP3, caspase-1 p20, pro-IL-1β, and IL-1β p17, and quantification of active caspase-1 and mature IL-1β in ECs treated with high-salt and transfected with Ctr siRNA or NLRP3 siRNA. d Immunoblot images of NLRP3, caspase-1 p20, pro-IL-1β, and IL-1β p17, and quantification of active caspase-1 and mature IL-1β in ECs treated with high-salt, treated with NAC. All data were presented as mean ± SEM, N ≥3. *p < 0.05

Article Snippet: Recombinant adenovirus that containing the full cDNA sequence of NFAT5 (Ad-NFAT5), were purchased from Cyagen (Guangzhou, China).

Techniques: Western Blot, Activity Assay, Transfection

Journal: Cell communication and signaling : CCS

Article Title: NFAT5 mediates hypertonic stress-induced atherosclerosis via activating NLRP3 inflammasome in endothelium.

doi: 10.1186/s12964-019-0406-7

Figure Lengend Snippet: Fig. 6 High-salt-elevated NFAT5 mediates transcription of NLRP3 and IL-1β in ECs. a-b mRNA and protein levels of NLRP3 in ECs treated with Adenovirus-null (Ad-null, 5 MOI) and Adenovirus-NFAT5 (Ad-NFAT5, 2 MOI or 5 MOI). c-d High-salt increases binding of NFAT5 to the promoter region of IL-1β. Diagram showing the region of the NFAT5 binding site upstream of the transcription start site (TSS) of NLRP3, and the regions that were used to analyze NFAT5 binding by ChIP. ChIP results are relative to 270 mosmol/kg. e-f mRNA and protein levels of NLRP3 in ECs treated with high-salt and transfected with Ctr siRNA or NFAT5 siRNA. g Protein secretion of IL-1β in ECs treated with high-salt and transfected with Ctr siRNA or NFAT5 siRNA. h High-salt increases binding of NFAT5 to the promoter region of IL-1β. i Protein levels of pro-IL-1β in ECs treated with high-salt and transfected with Ctr siRNA or NFAT5 siRNA. All data were presented as mean ± SEM, N ≥3. *p < 0.05

Article Snippet: Recombinant adenovirus that containing the full cDNA sequence of NFAT5 (Ad-NFAT5), were purchased from Cyagen (Guangzhou, China).

Techniques: Binding Assay, ChIP-chip, Transfection

Journal: Cell communication and signaling : CCS

Article Title: NFAT5 mediates hypertonic stress-induced atherosclerosis via activating NLRP3 inflammasome in endothelium.

doi: 10.1186/s12964-019-0406-7

Figure Lengend Snippet: Fig. 7 Schematic summarizes the mechanism that NLRP3 inflammasome activation in endothelium mediates hypertonic stress-induced atherosclerosis via NFAT5. Schematic illustration of the process. Stage i: Hypertonicity →NFAT5-dependent NLRP3 gene transcription →NLRP3 inflammasome activation. Stage ii: NLRP3 inflammasome activation →NFAT5-transcription-mediated pro-IL-1β →IL-1β secretion. Stage iii: IL-1β secretion →adhesive molecules →monocytes adhesion and infiltration. Stage iv: The activation of endothelial innate immunity promotes macrophage-driven foam cells and phenotype conversion of smooth muscle cells, contributing to the formation of atherosclerosis

Article Snippet: Recombinant adenovirus that containing the full cDNA sequence of NFAT5 (Ad-NFAT5), were purchased from Cyagen (Guangzhou, China).

Techniques: Activation Assay, Adhesive

Journal: Microorganisms

Article Title: Cyclodextrin Counteracts Coxsackievirus-Induced Cardiac Damage by Protecting Desmosome Integrity and Suppressing Proinflammatory Cytokine Expression

doi: 10.3390/microorganisms13102294

Figure Lengend Snippet: Cardiac-Specific Nfat5 -knockout Reduces Desmoplakin and Disrupts Desmosomes in Mice. ( A ) Immunohistochemistry (IHC) staining and quantification of nuclear factor of activated T cells 5 (NFAT5) in heart tissues from wild-type (WT) and Nfat5-knockout (KO) mice. Brown staining indicates NFAT5 expression (blue arrows), and purple staining represents nuclei. ( B ) Detection of NFAT5 protein by Western blot from above heart tissues. Quantification of band density was conducted by ImageJ ( n = 5). ( C ) IHC staining and quantification of desmoplakin (DSP) in heart tissues from WT and KO mice. Brown staining indicates DSP expression (blue arrows), and purple staining represents nuclei. Quantification of stained area was performed using ImageJ ( n = 5). ( D ) Detection of DSP protein by Western blot from above heart tissues. ( E ) Transmission electron microscopy (TEM) images of intercalated disk (ICD) structures in hearts from Coxsackievirus B3 (CVB3)-infected mouse hearts. WT mice were either sham-infected with saline or infected with CVB3 for 7 days ( n = 5 for each group). Heart samples were collected and processed for TEM to examine cardiomyocyte ultrastructure. Red circles highlight desmosomes, and red arrow indicates CVB3 particles. From each mouse, three randomly selected views (100 μm 2 each) of heart sections were imaged and desmosome numbers were quantified and statistically analyzed. ( F ) TEM images of ICD structures in hearts from non-infected WT and KO mice ( n = 5 for each group). Red circles highlight desmosomes. Statistical analysis was performed using Student’s t -test with Welch’s correction. Data are presented as mean ± SEM. * p < 0.05; ** p < 0.01.

Article Snippet: siRNAs targeting human NFAT5 (#sc-43968) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Knock-Out, Immunohistochemistry, Staining, Expressing, Western Blot, Transmission Assay, Electron Microscopy, Infection, Saline

Journal: Microorganisms

Article Title: Cyclodextrin Counteracts Coxsackievirus-Induced Cardiac Damage by Protecting Desmosome Integrity and Suppressing Proinflammatory Cytokine Expression

doi: 10.3390/microorganisms13102294

Figure Lengend Snippet: DSP is a Direct Transcriptional Target of NFAT5. ( A ) Immunofluorescence (IF) of NFAT5 in HeLa cells transfected with NFAT5-specific siRNA (siNFAT5) or scrambled control siRNA (siCtrl), followed by IF of NFAT5 protein. Green fluorescence indicates NFAT5, and blue fluorescence (DAPI) marks nuclei. Quantification of immunofluorescence density was performed by ImageJ. ( B ) IF of DSP in HeLa cells transfected with siNFAT5 and subjected to IF for desmoplakin. Green fluorescence indicates desmoplakin protein, and blue fluorescence indicates nuclei. ( C ) Western blot analyses of NFAT5 and DSP proteins using HeLa cells transfected with siNFAT5 or siCtrl. Quantification of Western blot density was performed by ImageJ. ( D ) Determination of DSP mRNA levels by RT-qPCR using total RNA extracted from HeLa cells transfected with siNFAT5 (left) or Nfat5-knockout mouse heart tissues (right). Statistical analysis was performed as in . Data are presented as mean ± SEM, n = 5. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: siRNAs targeting human NFAT5 (#sc-43968) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Immunofluorescence, Transfection, Control, Fluorescence, Western Blot, Quantitative RT-PCR, Knock-Out

Journal: Microorganisms

Article Title: Cyclodextrin Counteracts Coxsackievirus-Induced Cardiac Damage by Protecting Desmosome Integrity and Suppressing Proinflammatory Cytokine Expression

doi: 10.3390/microorganisms13102294

Figure Lengend Snippet: HPβCD Inhibits CVB3 Replication and Induces NFAT5 Expression. ( A ) Western blot analysis of CVB3 VP1 protein levels after HeLa cells were pretreated with HPβCD (0–5.0 mM) for 24 h and subsequently infected with CVB3 (MOI = 10) for 5 h. Quantification of Western blot analyses was performed by ImageJ. ( B ) Plaque assay to quantify infectious viral particles. Culture supernatants were collected from infected cells in ( A ) and serially diluted to 10 −6 . ( C ) Western blot analysis of NFAT5 protein levels from cell lysates in ( A ). ( D ) Quantification of NFAT5 and DSP mRNA levels by RT-qPCR using total RNA extracted from the same cell lysates in ( A ). Statistical analysis was performed as in . Data are presented as mean ± SEM ( n = 5). * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: siRNAs targeting human NFAT5 (#sc-43968) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Expressing, Western Blot, Infection, Plaque Assay, Quantitative RT-PCR

Journal: Microorganisms

Article Title: Cyclodextrin Counteracts Coxsackievirus-Induced Cardiac Damage by Protecting Desmosome Integrity and Suppressing Proinflammatory Cytokine Expression

doi: 10.3390/microorganisms13102294

Figure Lengend Snippet: IF to determine the HPβCD-induced upregulation of both NFAT5 and desmoplakin, and suppression of CVB3 replication. Virus- and sham-infected HeLa cells were treated with 0 mM or 5 mM HPβCD for 24 h, followed by IF to evaluate the expression of ( A ) NFAT5, ( B ) desmoplakin, ( C ) CVB3 VP1, and ( D ) CVB3 dsRNA. Green fluorescence indicates NFAT5 or desmoplakin, red fluorescence indicates VP1 or dsRNA, and blue fluorescence marks the nuclei. Quantification of immunofluorescence density was performed by ImageJ. Statistical analysis was performed as in . Data are presented as mean ± SEM, n = 5. * p < 0.05; nd indicates not significant.

Article Snippet: siRNAs targeting human NFAT5 (#sc-43968) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Virus, Infection, Expressing, Fluorescence, Immunofluorescence

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: Oil Red O (ORO)- stained regions from the aortae of N5fl/fl and N5(SMC)−/− mice fed an atherogenic diet for 25 weeks and appropriate regions from aortae of normal chow dietfed N5fl/fl and N5(SMC)−/− mice (see ORO staining) were Target Host Dilution Cat. no.; supplier Application Primary antibodies Anti- alpha- Tubulin Rabbit 1:10 2144; Cell Signaling Technology Wes Anti- HDAC1 Rabbit 1:10 NB100- 56340; Novus Biologicals Wes Anti- histone H3 Rabbit 1:1000 ab1791; Abcam Wes Anti- NFAT5 Mouse 1:10 SC- 398171; Santa Cruz Wes Anti- NFAT5 Rabbit 1:100 NB120- 3446; Novus Biologicals IF Anti- NFAT5 Rabbit 1:500 NB120- 3446; Novus Biologicals Wes Anti- VCP Mouse 1:50 ab11433; Abcam Wes Secondary antibodies Anti- Mouse HRP Goat – 042- 205; Bio- Techne Wes Anti- Rabbit IgG- Cy3 Donkey 1:100 711- 166- 152; Dianova IF Abbreviations: IF, immunofluorescence; Wes, capillary electrophoresis.

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: Oil Red O (ORO)- stained regions from the aortae of N5fl/fl and N5(SMC)−/− mice fed an atherogenic diet for 25 weeks and appropriate regions from aortae of normal chow dietfed N5fl/fl and N5(SMC)−/− mice (see ORO staining) were Target Host Dilution Cat. no.; supplier Application Primary antibodies Anti- alpha- Tubulin Rabbit 1:10 2144; Cell Signaling Technology Wes Anti- HDAC1 Rabbit 1:10 NB100- 56340; Novus Biologicals Wes Anti- histone H3 Rabbit 1:1000 ab1791; Abcam Wes Anti- NFAT5 Mouse 1:10 SC- 398171; Santa Cruz Wes Anti- NFAT5 Rabbit 1:100 NB120- 3446; Novus Biologicals IF Anti- NFAT5 Rabbit 1:500 NB120- 3446; Novus Biologicals Wes Anti- VCP Mouse 1:50 ab11433; Abcam Wes Secondary antibodies Anti- Mouse HRP Goat – 042- 205; Bio- Techne Wes Anti- Rabbit IgG- Cy3 Donkey 1:100 711- 166- 152; Dianova IF Abbreviations: IF, immunofluorescence; Wes, capillary electrophoresis.

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: Oil Red O (ORO)- stained regions from the aortae of N5fl/fl and N5(SMC)−/− mice fed an atherogenic diet for 25 weeks and appropriate regions from aortae of normal chow dietfed N5fl/fl and N5(SMC)−/− mice (see ORO staining) were Target Host Dilution Cat. no.; supplier Application Primary antibodies Anti- alpha- Tubulin Rabbit 1:10 2144; Cell Signaling Technology Wes Anti- HDAC1 Rabbit 1:10 NB100- 56340; Novus Biologicals Wes Anti- histone H3 Rabbit 1:1000 ab1791; Abcam Wes Anti- NFAT5 Mouse 1:10 SC- 398171; Santa Cruz Wes Anti- NFAT5 Rabbit 1:100 NB120- 3446; Novus Biologicals IF Anti- NFAT5 Rabbit 1:500 NB120- 3446; Novus Biologicals Wes Anti- VCP Mouse 1:50 ab11433; Abcam Wes Secondary antibodies Anti- Mouse HRP Goat – 042- 205; Bio- Techne Wes Anti- Rabbit IgG- Cy3 Donkey 1:100 711- 166- 152; Dianova IF Abbreviations: IF, immunofluorescence; Wes, capillary electrophoresis.

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: Oil Red O (ORO)- stained regions from the aortae of N5fl/fl and N5(SMC)−/− mice fed an atherogenic diet for 25 weeks and appropriate regions from aortae of normal chow dietfed N5fl/fl and N5(SMC)−/− mice (see ORO staining) were Target Host Dilution Cat. no.; supplier Application Primary antibodies Anti- alpha- Tubulin Rabbit 1:10 2144; Cell Signaling Technology Wes Anti- HDAC1 Rabbit 1:10 NB100- 56340; Novus Biologicals Wes Anti- histone H3 Rabbit 1:1000 ab1791; Abcam Wes Anti- NFAT5 Mouse 1:10 SC- 398171; Santa Cruz Wes Anti- NFAT5 Rabbit 1:100 NB120- 3446; Novus Biologicals IF Anti- NFAT5 Rabbit 1:500 NB120- 3446; Novus Biologicals Wes Anti- VCP Mouse 1:50 ab11433; Abcam Wes Secondary antibodies Anti- Mouse HRP Goat – 042- 205; Bio- Techne Wes Anti- Rabbit IgG- Cy3 Donkey 1:100 711- 166- 152; Dianova IF Abbreviations: IF, immunofluorescence; Wes, capillary electrophoresis.

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: Oil Red O (ORO)- stained regions from the aortae of N5fl/fl and N5(SMC)−/− mice fed an atherogenic diet for 25 weeks and appropriate regions from aortae of normal chow dietfed N5fl/fl and N5(SMC)−/− mice (see ORO staining) were Target Host Dilution Cat. no.; supplier Application Primary antibodies Anti- alpha- Tubulin Rabbit 1:10 2144; Cell Signaling Technology Wes Anti- HDAC1 Rabbit 1:10 NB100- 56340; Novus Biologicals Wes Anti- histone H3 Rabbit 1:1000 ab1791; Abcam Wes Anti- NFAT5 Mouse 1:10 SC- 398171; Santa Cruz Wes Anti- NFAT5 Rabbit 1:100 NB120- 3446; Novus Biologicals IF Anti- NFAT5 Rabbit 1:500 NB120- 3446; Novus Biologicals Wes Anti- VCP Mouse 1:50 ab11433; Abcam Wes Secondary antibodies Anti- Mouse HRP Goat – 042- 205; Bio- Techne Wes Anti- Rabbit IgG- Cy3 Donkey 1:100 711- 166- 152; Dianova IF Abbreviations: IF, immunofluorescence; Wes, capillary electrophoresis.

Techniques: Staining, Light Microscopy, Control, Confocal Microscopy

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: 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

Journal: Science advances

Article Title: Direct ionic stress sensing and mitigation by the transcription factor NFAT5.

doi: 10.1126/sciadv.adu3194

Figure Lengend Snippet: Fig. 1. Genetic screens to identify positive and negative regulators of the transcriptional response to hypertonic stress. (A) Temporal sequence of cellular changes triggered by hypertonic stress. (B) Expression of NFAT5 target genes after 8 hours in isotonic media (300 mOsm/liter) or hypertonic media [NaCl (+200 mOsm/liter), sorbi- tol, or urea]. (C) Expression of the NFAT5 target gene Akr1b3 in wild-type (WT) IMCD3 cells or a clonal Nfat5−/− cell line after 8 hours in isotonic or hypertonic media [NaCl (+200 mOsm/liter)]. See fig. S1A. (D) GFP fluorescence in IMCD3-G reporter cells stably carrying the 8xTonE-GFP transcriptional reporter (left) to measure NFAT5 activity after 8 hours in isotonic or hypertonic media (+200 mOsm/liter). Each point depicts the median GFP fluorescence from >2000 cells. (E) 8xTonE-GFP activity in IMCD3-G cells in response to increasing amounts of NaCl added to isotonic media. Each point shows the mean ± SD of three independent median measurements from >2000 cells. (F) 8xTonE-GFP activity after exposure to hypertonic media [NaCl (+200 mOsm/liter), 8 hours] in WT or Nfat5−/− IMCD3 cells. (G) Strategy for genome-wide loss-of-function screens in mouse IMCD3 and human HAP1 cells using a stably integrated 8xTonE-GFP reporter. See fig. S2A. (H) Results from the HAP1 screen outlined in (G). The x axis shows the Intronic Gene-trap Insertion Orientation Bias (IGTIOB) score (28), which scores the bias toward inactivating insertions in each gene, and the y axis shows the false discovery rate (FDR)–adjusted P value, reflecting the enrichment of gene trap (GT) insertions in sorted over unsorted cells. Statistics: Bars [(B) and (D)] or black hori- zontal lines [(C) and (F)] denote mean values calculated from independent measurements shown as points. Statistical significance was determined by a two-way analysis of variance (ANOVA) test with Sidak’s multiple comparisons posttest (n > 3). ****P < 0.0001, **P < 0.01, and *P < 0.05. See also figs. S1 and S2. ns, nonsignificant.

Article Snippet: Introduction of NFAT5 variants and the NFAT5 transcriptional reporter into yeast W303a cells were transformed with pAG306- 8xTonE- pCYC1- GFP, constructed by replacing the GPD1 promoter in Addgene plasmid #14140 (pAG306- GPD) with a 8xTonE- pCYC1- GFP cassette, which was adapted from a yeast transcriptional reporter for calcineurin activity (pAMS366- 4xCDRE- GFP- PEST, Addgene plasmid #138658) by replacing the four calcineurin- dependent response elements (CDREs) with eight copies of the TonE binding sequence (5′- TGGAAAATTAC- 3′) (82).

Techniques: Sequencing, Expressing, Fluorescence, Stable Transfection, Activity Assay, Genome Wide

Journal: Science advances

Article Title: Direct ionic stress sensing and mitigation by the transcription factor NFAT5.

doi: 10.1126/sciadv.adu3194

Figure Lengend Snippet: Fig. 2. NFAT5 can be activated by hypertonic stress in S. cerevisiae. (A) Domain structures of mVenus-tagged human full-length, nuclear, and mini variants of NFAT5. Nuclear NFAT5 was constitutively targeted to the nucleus by removal of its endogenous nuclear localization signal (NLS) and nuclear export signal (NES) sequences and addition of a strong foreign NLS. (B and C) Expression of an NFAT5 target gene in Nfat5−/− IMCD3 cells stably expressing NFAT5 variants [see (A)] in isotonic or hypertonic [NaCl (+200 mOsm/liter), 8 hours] media. See fig. S3A for protein abundances. (D to F) Structure of the 8xTonE-pCYC1-GFP reporter and galactose-inducible (pGAL1) mini- NFAT5 variant genes integrated into WT W303a yeast cells. Box (D) shows the workflow used to measure reporter activity after expression of mRuby3, mRuby3-DBD, or mRuby3-mini-NFAT5 (E) or exposure of cells expressing mRuby3-mini-NFAT5 to various solutes (F). All solutes were added at the indicated concentrations to complete synthetic media (CSM). (G and H) 8xTonE-pCYC1-GFP reporter activity (H) in yeast cells expressing DNA binding (DB) or dimerization (DIM) mutants of NLS-mRuby3-mini- NFAT5 (G). (I) Response of a mutant 8xTonE-pCYC1-GFP reporter (left) known be impaired in binding to NFAT5 to increasing hypertonic stress. (J) The HOG (high-osmolarity glycerol) pathway in S. cerevisiae. Colored X’s denote three different genes or gene sets that were deleted to disrupt the pathway at various levels: HOG1, PBS2, or the combined deletion of SSK2, SSK22, and SHO1. (K) 8xTonE-pCYC1-GFP reporter activity in WT, hog1Δ, pbs2Δ, or ssk2Δ ssk22Δ sho1Δ cells (also expressing mini-NFAT5). Statistics: Each point [(E), (F), (H), (I), and (K)] shows the mean ± SD of >3 median measurements, each from >5000 cells. Solid horizontal lines [(B) and (C)] denote mean values (n = 3). [(B) and (C)] Two-way ANOVA test with Sidak’s multiple comparisons posttest (n > 3). ****P < 0.0001 and *P < 0.05. See also fig. S3.

Article Snippet: Introduction of NFAT5 variants and the NFAT5 transcriptional reporter into yeast W303a cells were transformed with pAG306- 8xTonE- pCYC1- GFP, constructed by replacing the GPD1 promoter in Addgene plasmid #14140 (pAG306- GPD) with a 8xTonE- pCYC1- GFP cassette, which was adapted from a yeast transcriptional reporter for calcineurin activity (pAMS366- 4xCDRE- GFP- PEST, Addgene plasmid #138658) by replacing the four calcineurin- dependent response elements (CDREs) with eight copies of the TonE binding sequence (5′- TGGAAAATTAC- 3′) (82).

Techniques: Expressing, Stable Transfection, Variant Assay, Activity Assay, Binding Assay, Mutagenesis

Journal: Science advances

Article Title: Direct ionic stress sensing and mitigation by the transcription factor NFAT5.

doi: 10.1126/sciadv.adu3194

Figure Lengend Snippet: Fig. 3. NFAT5 is activated by ionic stress. (A) Mechanism of ammonium acetate (NH4OAc) permeation into cells. (B and C) Confocal images (xz plane) of IMCD3 cells exposed to NaCl or NH4OAc (+200 mOsm/liter) showing nuclei [4′,6-diamidino-2-phenylindole (DAPI), top] or the plasma membrane (CellMask, bottom). Cell heights (n > 28 per condition) calculated from such images are shown at various time points after solute addition (C). (D) Volume of single IMCD3 cells (n > 26 per condition) 10 min after the addition of various solutes (+200 mOsm/liter). (E) Change in the mean (±SEM, n = 20) fluorescence ratio from a genetically encoded ionic strength sensor (left) expressed in IMCD3 cells exposed to NH4OAc (200 mOsm/liter). (F and G) Distribution of GFP-WNK1 stably expressed in Wnk1−/− IMCD3 cells 30 min after the addition of various solutes (+400 mOsm/liter, 30 min). (G) Abundances of phosphorylated and total SPAK in IMCD3 cells 30 min after the addition of various solutes (+400 mOsm/ liter). (H) Nuclear mVenus-NFAT5 fluorescence (n > 145 per condition) in Nfat5−/−:mVenus-NFAT5 IMCD3 cells after the addition of NaCl or NH4OAc (+200 mOsm/liter). See fig. S4A. (I) Dose-response relationship between NH4OAc concentration and 8xTonE-GFP reporter activity. Each point shows the mean ± SD of three median measure- ments from >2000 cells. (J) 8xTonE-pCYC1-GFP reporter activity in yeast cells expressing mini-NFAT5 (Fig. 2D) in response to NH4OAc. Each point shows the mean ± SD of six median measurements from >5000 cells. (K) The diameter of yeast cells (n > 98 per condition) 5 min after the addition of NaCl (1200 mOsm/liter) or NH4OAc. Scale bars, 2 μm (B) and 10 μm (F). Statistics: (C), (D), (H), and (K) show single-cell measurements and the population median. Kruskal-Wallis test with Dunn’s multiple comparisons test. ****P < 0.0001, ***P < 0.001, and **P < 0.01. See also fig. S4.

Article Snippet: Introduction of NFAT5 variants and the NFAT5 transcriptional reporter into yeast W303a cells were transformed with pAG306- 8xTonE- pCYC1- GFP, constructed by replacing the GPD1 promoter in Addgene plasmid #14140 (pAG306- GPD) with a 8xTonE- pCYC1- GFP cassette, which was adapted from a yeast transcriptional reporter for calcineurin activity (pAMS366- 4xCDRE- GFP- PEST, Addgene plasmid #138658) by replacing the four calcineurin- dependent response elements (CDREs) with eight copies of the TonE binding sequence (5′- TGGAAAATTAC- 3′) (82).

Techniques: Clinical Proteomics, Membrane, Fluorescence, Stable Transfection, Concentration Assay, Activity Assay, Expressing

Journal: Science advances

Article Title: Direct ionic stress sensing and mitigation by the transcription factor NFAT5.

doi: 10.1126/sciadv.adu3194

Figure Lengend Snippet: Fig. 4. NFAT5 forms biomolecular condensates in cells exposed to hypertonic or ionic stress. (A) NFAT5 has a predicted prion-like domain (PLD) and a structured DBD embedded within IDRs (gray). (B and C) Snapshots from live cell imaging of HEK293T cells transiently transfected with GFP-NFAT5 and subjected to hypertonic stress [NaCl (+100 mOsm/liter)]. Mean (±SEM, n = 15) number of droplets per cell is shown on the graph (C, right) during a isotonic-hypertonic-isotonic stress cycle [(C), left]. (D) Snapshots (left) and recovery curve (right, mean ± SEM, n = 9) from a fluorescence recovery after photobleaching (FRAP) experiment on GFP-NFAT5 condensates in HEK293T subjected to hypertonic stress [NaCl (+100 mOsm/liter), 30 min]. (E and F) Subcellular distributions of full-length GFP-NFAT5 (E) or mVenus-mini-NFAT5 (F) stably expressed from a single locus in Nfat5−/− IMCD3 cells after the addition of various solutes (+200 mOsm/liter, 30 min). (G) Live cell time course of NH4OAc treated IMCD3 cells carrying NFAT5 tagged at its endogenous genomic locus with mNeonGreen (mNG) [see fig. S6 (C to E)]. In (E) to (G), line scans show fluorescence intensity traces along the trajectories of the yellow line in the images. (H and I) Endogenously tagged mNG-NFAT5 nuclear condensates in IMCD3 cells [see fig. S6 (C) to (E)] were imaged by structured illumination microscopy (H) and enumerated (I, n ~ 34 cells with median indicated) after the addition of NaCl or NH4OAc (+200 mOsm/liter, 30 min). Kruskal- Wallis test with Dunn’s multiple comparisons posttest. ****P < 0.0001. (J) FRAP images and recovery curve (n = 13; mean ± SEM) of endogenous mNG-NFAT5 puncta in IMCD3 cells subjected to ionic stress [NH4OAc (+200 mOsm/liter), 30 min]. Scale bars, 10 μm [(B), (C), (E), (F), (G), and (H)] and 1 μm [(D) and (J)]. See also figs. S5 and S6. a.u., arbitrary units.

Article Snippet: Introduction of NFAT5 variants and the NFAT5 transcriptional reporter into yeast W303a cells were transformed with pAG306- 8xTonE- pCYC1- GFP, constructed by replacing the GPD1 promoter in Addgene plasmid #14140 (pAG306- GPD) with a 8xTonE- pCYC1- GFP cassette, which was adapted from a yeast transcriptional reporter for calcineurin activity (pAMS366- 4xCDRE- GFP- PEST, Addgene plasmid #138658) by replacing the four calcineurin- dependent response elements (CDREs) with eight copies of the TonE binding sequence (5′- TGGAAAATTAC- 3′) (82).

Techniques: Live Cell Imaging, Transfection, Fluorescence, Stable Transfection, Microscopy

Journal: Science advances

Article Title: Direct ionic stress sensing and mitigation by the transcription factor NFAT5.

doi: 10.1126/sciadv.adu3194

Figure Lengend Snippet: Fig. 5. The PLD of NFAT5 is a sensor of solution ionic strength. (A) Distribution of GFP-tagged NFAT CTD (Fig. 2A) or PLD (Fig. 4A) in HEK293T cells exposed to various solutes (+100 mOsm/liter, 30 min). See fig. S7A. (B) Expression of an NFAT5 target gene (mean ± SD, n = 3) in Nfat5−/− IMCD3 cells stably expressing NFAT5 variants. See fig. S7B. (C and D) Domain structure and cellular distribution of GFP-tagged WNK1 and WNK1-NFAT5 chimera proteins. The WNK1 IDR [amino acids (a.a.) 495 to 2382], a sensor of macromolecular crowding, was replaced with the CTD or the PLD of NFAT5. Graphs (D) show the number of puncta per cell (n > 20 cells with median indicated). Kruskal-Wallis with Dunn’s multiple comparisons test, ****P < 0.0001. (E) Synthetic TFs (left) constructed from the DBD of GAL4 fused to the NFAT5 CTD, PLD or NTD (Figs. 2A and 4A) were tested for their abilities to activate a firefly luciferase reporter (n > 3, mean indicated) driven by GAL4 binding sites. Two-way ANOVA test and Sidak’s multiple comparisons test, ***P <0.001. (F) Fluorescence microscopy was used to assess condensate formation in vitro by purified (fig. S7C) GFP-CTD (70 μM, top row) and GFP-PLD (90 μM, bottom row). (G) Reversibility of GFP-CTD condensates assessed by a centrifugation and resuspension assay. All solutions contain 5% dextran. (H) Phase diagrams for purified GFP-CTD (left) and GFP-PLD (right). The boundary between the shaded and unshaded areas of the graph is taken as the phase boundary; crossing this boundary leads to the abrupt drop of diffuse fluorescence and emergence of droplets. Images were obtained across three replicates per condition. Scale bars, 10 μm [(A) and (D)] and 5 μm (F). See also fig. S7.

Article Snippet: Introduction of NFAT5 variants and the NFAT5 transcriptional reporter into yeast W303a cells were transformed with pAG306- 8xTonE- pCYC1- GFP, constructed by replacing the GPD1 promoter in Addgene plasmid #14140 (pAG306- GPD) with a 8xTonE- pCYC1- GFP cassette, which was adapted from a yeast transcriptional reporter for calcineurin activity (pAMS366- 4xCDRE- GFP- PEST, Addgene plasmid #138658) by replacing the four calcineurin- dependent response elements (CDREs) with eight copies of the TonE binding sequence (5′- TGGAAAATTAC- 3′) (82).

Techniques: Expressing, Stable Transfection, Construct, Luciferase, Binding Assay, Fluorescence, Microscopy, In Vitro, Purification, Centrifugation

Journal: Science advances

Article Title: Direct ionic stress sensing and mitigation by the transcription factor NFAT5.

doi: 10.1126/sciadv.adu3194

Figure Lengend Snippet: Fig. 6. NFAT5 activity correlates with phase separation propensity. (A) Distri- bution of GFP-NFAT5 (left) stably expressed in Nfat5−/− IMCD3 cells exposed to hy- pertonic stress [NaCl (+200 mOsm/liter)] in the presence of 1% 1,6-hexanediol (1,6-HD) or 2,5-hexanediol (2,5-HD). The graph on the right shows the percentage of cells with nuclear puncta (>100 cells per data point), with each bar depicting the mean of six to seven independent measurements. (B) Expression of the NFAT5 tar- get gene Akr1b3 in response to a 10-hour treatment of 1% 1,6-HD or 2,5-HD in iso- tonic or hypertonic [NaCl (+200 mOsm/liter)] media. Bars denote the mean of four independent experiments shown as points. (C) Vertical blue lines mark the position of the seven histidines within the PLD targeted for mutagenesis to phenylalanine (F) or lysine (K) in mini-NFAT5. (D) Distribution of mVenus fluorescence in the nucle- us of Nfat5−/− cells stably expressing the indicated variants of mini-NFAT5 after 30 min in isotonic or hypertonic [NaCl (+200 or +300 mOsm/liter)] media. (E) Number of mini-NFAT5 nuclear puncta per cell (top) and target gene expression (bottom) in IMCD3 cells treated for 8 hours with increasing concentrations of NaCl. Each point shows the mean ± SD of three independent experiments (n > 30 cells each). Scale bars, 10 μm [(A) and (D)]. Statistics: Statistical significance was determined by a two-way ANOVA test, Sidak’s multiple comparisons. ****P < 0.0001. See also figs. S8 and S9.

Article Snippet: Introduction of NFAT5 variants and the NFAT5 transcriptional reporter into yeast W303a cells were transformed with pAG306- 8xTonE- pCYC1- GFP, constructed by replacing the GPD1 promoter in Addgene plasmid #14140 (pAG306- GPD) with a 8xTonE- pCYC1- GFP cassette, which was adapted from a yeast transcriptional reporter for calcineurin activity (pAMS366- 4xCDRE- GFP- PEST, Addgene plasmid #138658) by replacing the four calcineurin- dependent response elements (CDREs) with eight copies of the TonE binding sequence (5′- TGGAAAATTAC- 3′) (82).

Techniques: Activity Assay, Stable Transfection, Expressing, Mutagenesis, Fluorescence, Targeted Gene Expression

Journal: Science advances

Article Title: Direct ionic stress sensing and mitigation by the transcription factor NFAT5.

doi: 10.1126/sciadv.adu3194

Figure Lengend Snippet: Fig. 7. NFAT5 condensates recruit transcriptional coactivators. (A to C) Recruitment of MED1 (A), BRD4 (B), and Pol II (C) in stress-induced nuclear NFAT5 condensates in Nfat5−/− IMCD3 cells stably expressing GFP-NFAT5 after the addition of NaCl or NH4OAc (+200 mOsm/liter, 30 min). Line scans show fluorescence intensity traces for NFAT5 (gray) and the second protein (dark teal) along the trajectories of the yellow line in the images. Overlapping peaks in these traces indicate colocalized puncta, which are also highlighted in the zoomed insets. (D) Cells were treated (left diagram) with increasing concentrations of dBET6 to acutely induce BRD4 degradation (assessed by the immunoblot on the right). (E) Impact of dBET6 degradation [as shown in (D)] on induction of two NFAT5 target genes (Slc6a12 and Akr1b3) in response to hypertonic stress [NaCl (+200 mOsm/liter)] for 11 hours. Bars denote the mean of three measurements, and the experiment was repeated three times. Scale bars, 10 μm [(A) to (C)]. See also figs. S12 and S13.

Article Snippet: Introduction of NFAT5 variants and the NFAT5 transcriptional reporter into yeast W303a cells were transformed with pAG306- 8xTonE- pCYC1- GFP, constructed by replacing the GPD1 promoter in Addgene plasmid #14140 (pAG306- GPD) with a 8xTonE- pCYC1- GFP cassette, which was adapted from a yeast transcriptional reporter for calcineurin activity (pAMS366- 4xCDRE- GFP- PEST, Addgene plasmid #138658) by replacing the four calcineurin- dependent response elements (CDREs) with eight copies of the TonE binding sequence (5′- TGGAAAATTAC- 3′) (82).

Techniques: Stable Transfection, Expressing, Fluorescence, Western Blot

Journal: Science advances

Article Title: Direct ionic stress sensing and mitigation by the transcription factor NFAT5.

doi: 10.1126/sciadv.adu3194

Figure Lengend Snippet: Fig. 8. Ionic stress response regulation by the NFAT5 PLD. (A) Position of the four 300–amino acid fragments of NFAT5 tested in (B) to (D). (B) Recruitment of endoge- nous BRD4 (red) to a TetO array in U2OS cells by EGFP-TetR DBD (green) fused to the four fragments of NFAT5 (see fig. S12A). Insets show a magnified view of the TetO array, visualized as a single dot of EGFP fluorescence. Enrichment of BRD4 in the EGFP-marked TetO array is plotted on the right for individual cells, with the mean indicated. Scale bars, 10 μm. (C) Condensate formation by hemagglutinin-tagged NFAT5 fragments in HEK293T cells (n > 25, median indicated). (D) Transactivation capacity of NFAT5 fragments (n = 3, bars show mean) or the VP16 AD (as a control) using the reporter assay shown in Fig. 5E. (E) A model for hypertonic and ionic stress adaptation. The IDR in WNK1 and PLD in NFAT5 each sense specific chemical properties of the intracellular environment. In response to hypertonic stress, the rapid loss of cell volume leads to an increase in macromolecular crowding, which activates the crowding sensor kinase WNK1 (but not NFAT5) (9). Through a kinase cascade, WNK1 activates transporters that increase intracellular ion concentrations, allowing cytoplasmic rehydration and volume recovery at the expense of elevated ionic strength. If persistent, this increase in ionic strength is the trigger for NFAT5 activation, leading to a transcriptional response that exchanges these ions for osmolytes. We speculate that NFAT5 has evolved to sense and facilitate adaptation to diverse ionic stressors (even those, like NH4OAc, that do not cause hypertonic stress). Statistics: Statistical significance was determined by a Kruskal-Wallis test, Dunn’s multiple comparisons [(B) and (C)], or a two-way ANOVA with Sidak’s multiple comparisons test (D). ****P < 0.0001 and **P < 0.01. See also figs. S12 and S13.

Article Snippet: Introduction of NFAT5 variants and the NFAT5 transcriptional reporter into yeast W303a cells were transformed with pAG306- 8xTonE- pCYC1- GFP, constructed by replacing the GPD1 promoter in Addgene plasmid #14140 (pAG306- GPD) with a 8xTonE- pCYC1- GFP cassette, which was adapted from a yeast transcriptional reporter for calcineurin activity (pAMS366- 4xCDRE- GFP- PEST, Addgene plasmid #138658) by replacing the four calcineurin- dependent response elements (CDREs) with eight copies of the TonE binding sequence (5′- TGGAAAATTAC- 3′) (82).

Techniques: Fluorescence, Control, Reporter Assay, Activation Assay

Journal: Frontiers in Neurology

Article Title: Induction of Osmolyte Pathways in Skeletal Muscle Inflammation: Novel Biomarkers for Myositis

doi: 10.3389/fneur.2018.00846

Figure Lengend Snippet: Messenger RNA levels in cultured CCL-136 cells treated for 24 h.

Article Snippet: RNA concentration was measured with a Nanodrop 1000 (ThermoFisher Scientific). cDNA was prepared from 200 ng of RNA with SuperScript II reverse transcriptase, 500 ng/μl oligo dTs, 0.1 M DTT, and 10 mM dNTPs each (Invitrogen, Darmstadt, Germany). cDNA was quantified through PCR reaction with Taqman Gene Expression Master mix (Applied Biosystems, Foster City, CA), using 6-carboxy-fluorescein-labeled probes and specific primers for: SLC5A3, Hs00272857_s1; SLC6A6, Hs00161778_m1; SLC6A12, HS00758246_m1; AKR1B1, Hs00739326_m1; NFAT5, Hs00232437_m1; MAPK14, Hs01051152_m1; RELA, Hs00153294_m1; NFKB1, Hs00765730_m1; NFKB2, Hs01028901_g1; glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Hs99999905_m1 (Applied Biosystems).

Techniques: Cell Culture

Journal: Frontiers in Neurology

Article Title: Induction of Osmolyte Pathways in Skeletal Muscle Inflammation: Novel Biomarkers for Myositis

doi: 10.3389/fneur.2018.00846

Figure Lengend Snippet: Messenger RNA levels in cultured normal human myotubes treated for 24 h.

Article Snippet: RNA concentration was measured with a Nanodrop 1000 (ThermoFisher Scientific). cDNA was prepared from 200 ng of RNA with SuperScript II reverse transcriptase, 500 ng/μl oligo dTs, 0.1 M DTT, and 10 mM dNTPs each (Invitrogen, Darmstadt, Germany). cDNA was quantified through PCR reaction with Taqman Gene Expression Master mix (Applied Biosystems, Foster City, CA), using 6-carboxy-fluorescein-labeled probes and specific primers for: SLC5A3, Hs00272857_s1; SLC6A6, Hs00161778_m1; SLC6A12, HS00758246_m1; AKR1B1, Hs00739326_m1; NFAT5, Hs00232437_m1; MAPK14, Hs01051152_m1; RELA, Hs00153294_m1; NFKB1, Hs00765730_m1; NFKB2, Hs01028901_g1; glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Hs99999905_m1 (Applied Biosystems).

Techniques: Cell Culture

Journal: Frontiers in Neurology

Article Title: Induction of Osmolyte Pathways in Skeletal Muscle Inflammation: Novel Biomarkers for Myositis

doi: 10.3389/fneur.2018.00846

Figure Lengend Snippet: Messenger RNA levels in cultured normal human myotubes treated with cytokines or added NaCl for up to 3 days.

Article Snippet: RNA concentration was measured with a Nanodrop 1000 (ThermoFisher Scientific). cDNA was prepared from 200 ng of RNA with SuperScript II reverse transcriptase, 500 ng/μl oligo dTs, 0.1 M DTT, and 10 mM dNTPs each (Invitrogen, Darmstadt, Germany). cDNA was quantified through PCR reaction with Taqman Gene Expression Master mix (Applied Biosystems, Foster City, CA), using 6-carboxy-fluorescein-labeled probes and specific primers for: SLC5A3, Hs00272857_s1; SLC6A6, Hs00161778_m1; SLC6A12, HS00758246_m1; AKR1B1, Hs00739326_m1; NFAT5, Hs00232437_m1; MAPK14, Hs01051152_m1; RELA, Hs00153294_m1; NFKB1, Hs00765730_m1; NFKB2, Hs01028901_g1; glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Hs99999905_m1 (Applied Biosystems).

Techniques: Cell Culture