Journal: International Journal of Biological Sciences

Article Title: NFAT5 is Regulated by p53/miR-27a Signal Axis and Promotes Mouse Ovarian Granulosa Cells Proliferation

doi: 10.7150/ijbs.29273

Figure Lengend Snippet: Identification of NFAT5 gene as a direct target of miR-27a in CHO-K1 cells. (A) Binding sites for miR-27a in the 3ˈ-UTR of NFAT5 gene predicted by TargetScan. Red characters indicate sequences that were mutated to abolish the interaction between miR-27a and the NFAT5 3ˈ-UTR. (B) The miR-27a binding site (Red) of different species in the NFAT5 3ˈ-UTR. (C) miR-27a mimics were transfected into CHO-K1 cells together with the pmirGLO-NFAT5 and pmirGLO-NFAT5-mut plasmid. Mimics NC was used as a negative control. (D) Endogenous NFAT5 mRNA levels were detected 24 h after CHO-K1 cells were transfected with mimics, mimics NC, inhibitor and inhibitor NC of miR-27a. (E) Western blot analysis was used to detect endogenous NFAT5 protein expression level 48 h after CHO-K1 cells were transfected with mimics, mimics NC, inhibitor and inhibitor NC of miR-27a. (F) Endogenous NFAT5 mRNA levels were detected 24 h after CHO-K1 cells were transfected with pc-p53 , pcDNA3.1 , si-p53 or NC. (G) Western blot analysis was used to detect endogenous NFAT5 protein expression level 48 h after CHO-K1 cells were transfected with pc-p53 , pcDNA3.1 , si-p53 or NC.The data are represented as mean ± S.D. of three independent experiments. ** P < 0.01, *** P < 0.001.

Article Snippet: Then, the membranes were blocked with skim milk and probed with Anti-p53 (A0263, abclonal, USA), Anti-NFAT5 (Bs-9473R, bioss,China), Anti-β-catenin (Ab32572, Abcam, USA), Anti-c-myc (Ab32072, Abcam, USA), Anti-Bcl-2 (12789-1-ap, Proteintech, China). β-actin (ab8226, Santa Cruz, USA) was used as a loading control.

Techniques: Binding Assay, Transfection, Plasmid Preparation, Negative Control, Western Blot, Expressing

Journal: International Journal of Biological Sciences

Article Title: NFAT5 is Regulated by p53/miR-27a Signal Axis and Promotes Mouse Ovarian Granulosa Cells Proliferation

doi: 10.7150/ijbs.29273

Figure Lengend Snippet: NFAT5 regulated the downstream genes of the Wnt signaling pathway. CHO-K1 cells were transfected with pc-NFAT5 , pcDNA3.1 and si-NFAT5, NC. (A) qRT-PCR was used to detect endogenous NFAT5 mRNA 24 h after transfection. (B) Western blot analysis was used to detect NFAT5 protein expression levels 48 h after transfection. (C, D) Twenty-four hours after transfection, the expression levels of β-catenin and Bcl-2 were determined by qRT-PCR. (E) Western blot analysis was used to detect endogenous β-catenin and Bcl-2 protein expression level. The data are represented as mean±S.D. of three independent experiments. * P < 0.05, *** P < 0.001.

Article Snippet: Then, the membranes were blocked with skim milk and probed with Anti-p53 (A0263, abclonal, USA), Anti-NFAT5 (Bs-9473R, bioss,China), Anti-β-catenin (Ab32572, Abcam, USA), Anti-c-myc (Ab32072, Abcam, USA), Anti-Bcl-2 (12789-1-ap, Proteintech, China). β-actin (ab8226, Santa Cruz, USA) was used as a loading control.

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Expressing

Journal: International Journal of Biological Sciences

Article Title: NFAT5 is Regulated by p53/miR-27a Signal Axis and Promotes Mouse Ovarian Granulosa Cells Proliferation

doi: 10.7150/ijbs.29273

Figure Lengend Snippet: Mouse GCs apoptosis induced by NFAT5 gene. (A) Mouse GCs were labeled with EdU (Red). Cell nuclei were also visualized by Hoechst staining (Blue). The images were representative of the results obtained. (B) The percentage of proliferating GCs was expressed as the ratio of EdU cells to total Hoechst cells. Data were presented as mean ± S.D. from nine independent experiments. *** P < 0.001.

Article Snippet: Then, the membranes were blocked with skim milk and probed with Anti-p53 (A0263, abclonal, USA), Anti-NFAT5 (Bs-9473R, bioss,China), Anti-β-catenin (Ab32572, Abcam, USA), Anti-c-myc (Ab32072, Abcam, USA), Anti-Bcl-2 (12789-1-ap, Proteintech, China). β-actin (ab8226, Santa Cruz, USA) was used as a loading control.

Techniques: Labeling, Staining

Journal: International Journal of Biological Sciences

Article Title: NFAT5 is Regulated by p53/miR-27a Signal Axis and Promotes Mouse Ovarian Granulosa Cells Proliferation

doi: 10.7150/ijbs.29273

Figure Lengend Snippet: A schematic illustration of the proposed model depicting a p53/miR-27a/NFAT5 mediated regulatory pathway in mouse GCs.

Article Snippet: Then, the membranes were blocked with skim milk and probed with Anti-p53 (A0263, abclonal, USA), Anti-NFAT5 (Bs-9473R, bioss,China), Anti-β-catenin (Ab32572, Abcam, USA), Anti-c-myc (Ab32072, Abcam, USA), Anti-Bcl-2 (12789-1-ap, Proteintech, China). β-actin (ab8226, Santa Cruz, USA) was used as a loading control.

Techniques:

Journal: International Journal of Biological Sciences

Article Title: NFAT5 is Regulated by p53/miR-27a Signal Axis and Promotes Mouse Ovarian Granulosa Cells Proliferation

doi: 10.7150/ijbs.29273

Figure Lengend Snippet: Primer sequences

Article Snippet: Then, the membranes were blocked with skim milk and probed with Anti-p53 (A0263, abclonal, USA), Anti-NFAT5 (Bs-9473R, bioss,China), Anti-β-catenin (Ab32572, Abcam, USA), Anti-c-myc (Ab32072, Abcam, USA), Anti-Bcl-2 (12789-1-ap, Proteintech, China). β-actin (ab8226, Santa Cruz, USA) was used as a loading control.

Techniques: Sequencing

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: 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: Expression of the mNG- NFAT5 fusion protein was confirmed by immunoblotting using an antibody against NFAT5 (Bethyl Laboratories) (fig. S6D).

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: Expression of the mNG- NFAT5 fusion protein was confirmed by immunoblotting using an antibody against NFAT5 (Bethyl Laboratories) (fig. S6D).

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: Expression of the mNG- NFAT5 fusion protein was confirmed by immunoblotting using an antibody against NFAT5 (Bethyl Laboratories) (fig. S6D).

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: Expression of the mNG- NFAT5 fusion protein was confirmed by immunoblotting using an antibody against NFAT5 (Bethyl Laboratories) (fig. S6D).

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: Expression of the mNG- NFAT5 fusion protein was confirmed by immunoblotting using an antibody against NFAT5 (Bethyl Laboratories) (fig. S6D).

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: Expression of the mNG- NFAT5 fusion protein was confirmed by immunoblotting using an antibody against NFAT5 (Bethyl Laboratories) (fig. S6D).

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: Expression of the mNG- NFAT5 fusion protein was confirmed by immunoblotting using an antibody against NFAT5 (Bethyl Laboratories) (fig. S6D).

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: Expression of the mNG- NFAT5 fusion protein was confirmed by immunoblotting using an antibody against NFAT5 (Bethyl Laboratories) (fig. S6D).

Techniques: Fluorescence, Control, Reporter Assay, Activation 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: 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

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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

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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

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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

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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