Journal: Science Advances

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

doi: 10.1126/sciadv.adu3194

Figure Lengend Snippet: ( 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), sorbitol, 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 , 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 horizontal 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: ( 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, hog1 Δ, pbs2 Δ, or ssk 2Δ ssk22 Δ sho1 Δ 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: ( A ) Mechanism of ammonium acetate (NH 4 OAc) permeation into cells. ( B and C ) Confocal images ( xz plane) of IMCD3 cells exposed to NaCl or NH 4 OAc (+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 NH 4 OAc (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 NH 4 OAc (+200 mOsm/liter). See fig. S4A. ( I ) Dose-response relationship between NH 4 OAc concentration and 8xTonE-GFP reporter activity. Each point shows the mean ± SD of three median measurements from >2000 cells. ( J ) 8xTonE- pCYC1 -GFP reporter activity in yeast cells expressing mini-NFAT5 in response to NH 4 OAc. 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 NH 4 OAc. 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: ( 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 NH 4 OAc 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 NH 4 OAc (+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 [NH 4 OAc (+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: ( A ) Distribution of GFP-tagged NFAT CTD or PLD 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 ( and ) 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: ( A ) Distribution of GFP-NFAT5 (left) stably expressed in Nfat5 −/− IMCD3 cells exposed to hypertonic 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 target gene Akr1b3 in response to a 10-hour treatment of 1% 1,6-HD or 2,5-HD in isotonic 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 nucleus 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: 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: ( 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 NH 4 OAc (+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: ( A ) Position of the four 300–amino acid fragments of NFAT5 tested in (B) to (D). ( B ) Recruitment of endogenous 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 . ( 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) . 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 NH 4 OAc, 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: Communications Biology

Article Title: HES1 potentiates high salt stress response as an enhancer of NFAT5-DNA binding

doi: 10.1038/s42003-024-06997-7

Figure Lengend Snippet: a , b A genome-wide siRNA screen to search for regulators of NFAT5 nuclear translocation under high salinity/hyperosmotic stress, 500 mOsm; 3 h. The dash line presented the value of NFAT5 nuclear translocation in non-target control siRNA-treated cells. b Notch-related genes in the positive hits whose knockdown attenuated NFAT5 nuclear translocation more than c-Abl1 knockdown. The vertical line shows the NFAT5 TL ratio, and the horizontal line shows the rank of genes. c KEGG pathway analysis of the 1291 positive hits. d , e Effect of HES1 depletion on NFAT5 nuclear translocation. Representative images of NFAT5-ΔtdTomato in the HeLa cells treated with siRNAs under ISO and high NaCl, 500 mOsm; 3 h. Blue and green lines represent the borders of the nuclear and whole-cell regions, respectively. The scale bar represents 10 µm. NT Non-transfected control. e NFAT5 TL ratio calculated from the fluorescent images ( n = 3, analyzing 400–800 cells in each experiment). f , g Effect of HES1 depletion on the subcellular distribution of endogenous NFAT5 in HeLa cells under ISO and high NaCl, 400 mOsm; 3 h. f LAMIN A/C and β-tubulin are nuclear and cytoplasmic marker proteins, respectively. NFAT5 TL ratio calculated from the band intensity ( g , n = 3). In the bar graphs, individual values (white points) and the mean ± SEM are presented. * p < 0.05, *** p < 0.001. ISO, 300 mOsm; high NaCl, 500 mOsm in ( d , e ) and 400 mOsm in ( f , g ); 3 h. See also Supplementary Fig. .

Article Snippet: The following antibodies were used for immunoblots(IB) or immunoprecipitation(IP) or immunofluorescence(IF) or Chromatin-IP(ChIP): FLAG antibody (1E6) (IB, Wako Pure Chemicals Industries, #014-22383); NFAT5 antibody (IB, IF, Santa Cruz Biotechnology, sc-13035; ChIP, Santa Cruz Biotechnology, sc-398171); HES1 antibody (IB, ChIP, Abcam, ab196328); LAMIN A/C antibody (IB, Cell Signaling Technology, #4777); beta-tubulin antibody (IB, Santa Cruz Biotechnology, sc-5274); alpha-tubulin antibody (IB, Santa Cruz Biotechnology, sc-53029); p44/42 MAPK (ERK1/2) antibody (IB, Cell Signaling Technology, #9102); phospho-p44/42 MAPK (ERK1/2) antibody (IB, Cell Signaling Technology, #9106); c-Myc antibody (IB, Sigma-Aldrich, A3853); Notch1 antibody (IB, Cell Signaling Technology, #4380); Cleaved Notch1 (Val1744) antibody (IB, Cell Signaling Technology, #4147); MEK1/2 antibody (IB, Cell Signaling Technology, #9122); DsRed antibody (IB, Santa Cruz Biotechnology, sc-101526); Anti-rebbit IgG, HRP-linked Antibody (IB, Cell Signaling Technology, #7074); Anti-mouse IgG, HRP-linked Antibody (IB, Cell Signaling Technology, #7076); Anti-rat IgG, HRP-linked Antibody (IB, Cell Signaling Technology, #7077).

Techniques: Genome Wide, Translocation Assay, Control, Knockdown, Transfection, Marker

Journal: Communications Biology

Article Title: HES1 potentiates high salt stress response as an enhancer of NFAT5-DNA binding

doi: 10.1038/s42003-024-06997-7

Figure Lengend Snippet: a Time course of HES1 protein expression in HeLa cells under ISO and high NaCl conditions, 400 mOsm. The bar graph indicates HES1 protein amount at 6 h after high NaCl stimuli ( n = 4). b mRNA expression of Hes1 in HeLa cells at 5 h after a high NaCl stimulus, 400 mOsm ( n = 5). mRNA expression in each sample was normalized to the mean expression of samples under isoosmotic conditions. c , d Effect of the ERK signaling inhibitor U0126 on high salt stress-induced HES1 protein under ISO and high NaCl conditions, 400 mOsm. Cells were pretreated with 5 µM U0126 for 30 min. DMSO: dimethyl sulfoxide. The bar graph indicates HES1 protein amount at 6 h after osmotic stimuli ( c , n = 3) and mRNA expression ( d , n = 3). mRNA expression in each sample was normalized to the mean expression of samples with DMSO treatment under isoosmotic conditions. e , f Effect of exogenous HES1 expression on NFAT5 nuclear translocation at 3 h after ISO and high NaCl stimuli, 400 mOsm. Representative images of NFAT5Δ-tdTomato in HeLa cells under isoomolarity. The scale bar represents 10 µm. EV: empty vector, a negative transfection control ( e ). NFAT5 TL ratio calculated from the fluorescent images ( f , n = 3–5, analyzing 40–80 cells in each experiment). In the bar graphs, individual values (white points) and the mean ± SEM are presented. * p < 0.05, ** p < 0.01, *** p < 0.001. ISO, 300 mOsm; high NaCl, 400 mOsm. See also Supplementary Fig. .

Article Snippet: The following antibodies were used for immunoblots(IB) or immunoprecipitation(IP) or immunofluorescence(IF) or Chromatin-IP(ChIP): FLAG antibody (1E6) (IB, Wako Pure Chemicals Industries, #014-22383); NFAT5 antibody (IB, IF, Santa Cruz Biotechnology, sc-13035; ChIP, Santa Cruz Biotechnology, sc-398171); HES1 antibody (IB, ChIP, Abcam, ab196328); LAMIN A/C antibody (IB, Cell Signaling Technology, #4777); beta-tubulin antibody (IB, Santa Cruz Biotechnology, sc-5274); alpha-tubulin antibody (IB, Santa Cruz Biotechnology, sc-53029); p44/42 MAPK (ERK1/2) antibody (IB, Cell Signaling Technology, #9102); phospho-p44/42 MAPK (ERK1/2) antibody (IB, Cell Signaling Technology, #9106); c-Myc antibody (IB, Sigma-Aldrich, A3853); Notch1 antibody (IB, Cell Signaling Technology, #4380); Cleaved Notch1 (Val1744) antibody (IB, Cell Signaling Technology, #4147); MEK1/2 antibody (IB, Cell Signaling Technology, #9122); DsRed antibody (IB, Santa Cruz Biotechnology, sc-101526); Anti-rebbit IgG, HRP-linked Antibody (IB, Cell Signaling Technology, #7074); Anti-mouse IgG, HRP-linked Antibody (IB, Cell Signaling Technology, #7076); Anti-rat IgG, HRP-linked Antibody (IB, Cell Signaling Technology, #7077).

Techniques: Expressing, Translocation Assay, Plasmid Preparation, Transfection, Control

Journal: Communications Biology

Article Title: HES1 potentiates high salt stress response as an enhancer of NFAT5-DNA binding

doi: 10.1038/s42003-024-06997-7

Figure Lengend Snippet: a , b Effect of HES1 depletion on the NFAT5 target genes BGT1 ( a , n = 3) and Aldose Reductase ( AR ) ( b , n = 5) at 7.5 h after ISO and high NaCl stimuli, 400 mOsm. mRNA expression in each sample was normalized to the mean expression of samples with control siRNA under isoosmotic conditions. c Expression of HES1 and NFAT5 in the HeLa cells transiently transfected with the EV or FLAG-HES1 construct at 6 h after ISO and high NaCl stimuli, 400 mOsm. d , e Effect of exogenous HES1 expression on the NFAT5 target genes BGT1 ( d , n = 4) and AR ( e , n = 4). f Endogenous HES1 expression in the HeLa cells transfected with a constitutively active Notch1 (NotchΔE-6Myc) construct at 6 h after ISO and high NaCl stimuli, 400 mOsm. g , h Effect of exogenous NotchΔE expression on the NFAT5 target genes BGT1 ( g , n = 3) and AR ( h , n = 3) at 7.5 h after ISO and high NaCl stimuli, 400 mOsm. In the bar graphs, individual values (white points) and the mean ± SEM are presented. N.S., not significant; * p < 0.05, ** p < 0.01, *** p < 0.001. ISO, 300 mOsm; high NaCl, 400 mOsm. See also Supplementary Fig. .

Article Snippet: The following antibodies were used for immunoblots(IB) or immunoprecipitation(IP) or immunofluorescence(IF) or Chromatin-IP(ChIP): FLAG antibody (1E6) (IB, Wako Pure Chemicals Industries, #014-22383); NFAT5 antibody (IB, IF, Santa Cruz Biotechnology, sc-13035; ChIP, Santa Cruz Biotechnology, sc-398171); HES1 antibody (IB, ChIP, Abcam, ab196328); LAMIN A/C antibody (IB, Cell Signaling Technology, #4777); beta-tubulin antibody (IB, Santa Cruz Biotechnology, sc-5274); alpha-tubulin antibody (IB, Santa Cruz Biotechnology, sc-53029); p44/42 MAPK (ERK1/2) antibody (IB, Cell Signaling Technology, #9102); phospho-p44/42 MAPK (ERK1/2) antibody (IB, Cell Signaling Technology, #9106); c-Myc antibody (IB, Sigma-Aldrich, A3853); Notch1 antibody (IB, Cell Signaling Technology, #4380); Cleaved Notch1 (Val1744) antibody (IB, Cell Signaling Technology, #4147); MEK1/2 antibody (IB, Cell Signaling Technology, #9122); DsRed antibody (IB, Santa Cruz Biotechnology, sc-101526); Anti-rebbit IgG, HRP-linked Antibody (IB, Cell Signaling Technology, #7074); Anti-mouse IgG, HRP-linked Antibody (IB, Cell Signaling Technology, #7076); Anti-rat IgG, HRP-linked Antibody (IB, Cell Signaling Technology, #7077).

Techniques: Expressing, Control, Transfection, Construct

Journal: Communications Biology

Article Title: HES1 potentiates high salt stress response as an enhancer of NFAT5-DNA binding

doi: 10.1038/s42003-024-06997-7

Figure Lengend Snippet: a Schematic model showing the design of the microarray analysis and gene classification. mRNA expression in HeLa cells treated with siControl, siNFAT5 and siHES1were analyzed at 7.5 h after ISO and high NaCl stimuli, 400 mOsm. b Heatmap showing the log fold change in the mRNA expression of high salt stress-induced genes at 7.5 h after a high NaCl stimulus, 400 mOsm. The expression under high salt conditions with each siRNA was compared with that under isoosmotic condition with siControl. c KEGG pathway analysis of the both NFAT5- and HES1-dependent genes (81 genes) in the microarray analysis. Effect of HES1 depletion on the both NFAT5- and HES1-dependent genes identified by microarray analysis at 7.5 h after ISO and high NaCl stimuli, 400 mOsm; ARG2 ( d , n = 4), GLS ( e , n = 5) and SLC2A1 ( f , n = 5). In the bar graphs, individual values (white points) and the mean ± SEM are presented. N.S. not significant; * p < 0.05, ** p < 0.01, *** p < 0.001. ISO, 300 mOsm; high NaCl, 400 mOsm. See also Supplementary Fig. .

Article Snippet: The following antibodies were used for immunoblots(IB) or immunoprecipitation(IP) or immunofluorescence(IF) or Chromatin-IP(ChIP): FLAG antibody (1E6) (IB, Wako Pure Chemicals Industries, #014-22383); NFAT5 antibody (IB, IF, Santa Cruz Biotechnology, sc-13035; ChIP, Santa Cruz Biotechnology, sc-398171); HES1 antibody (IB, ChIP, Abcam, ab196328); LAMIN A/C antibody (IB, Cell Signaling Technology, #4777); beta-tubulin antibody (IB, Santa Cruz Biotechnology, sc-5274); alpha-tubulin antibody (IB, Santa Cruz Biotechnology, sc-53029); p44/42 MAPK (ERK1/2) antibody (IB, Cell Signaling Technology, #9102); phospho-p44/42 MAPK (ERK1/2) antibody (IB, Cell Signaling Technology, #9106); c-Myc antibody (IB, Sigma-Aldrich, A3853); Notch1 antibody (IB, Cell Signaling Technology, #4380); Cleaved Notch1 (Val1744) antibody (IB, Cell Signaling Technology, #4147); MEK1/2 antibody (IB, Cell Signaling Technology, #9122); DsRed antibody (IB, Santa Cruz Biotechnology, sc-101526); Anti-rebbit IgG, HRP-linked Antibody (IB, Cell Signaling Technology, #7074); Anti-mouse IgG, HRP-linked Antibody (IB, Cell Signaling Technology, #7076); Anti-rat IgG, HRP-linked Antibody (IB, Cell Signaling Technology, #7077).

Techniques: Microarray, Expressing

Journal: Communications Biology

Article Title: HES1 potentiates high salt stress response as an enhancer of NFAT5-DNA binding

doi: 10.1038/s42003-024-06997-7

Figure Lengend Snippet: Schematic models showing the promoter regions of the NFAT5 target genes BGT1 ( a ) and Aldose Reductase (AR) ( b ). Each promoter region has two NFAT5-binding motifs, (i) and (ii), presented as white boxes. c – f ChIP analysis investigating the effect of HES1 depletion on the DNA binding of NFAT5 to each motif, BGT1-(i) and –(ii) and AR-(i) and –(ii) at 7.5 h after ISO and high NaCl stimuli, 400 mOsm ( n = 4). g Coimmunoprecipitation assay between over expressed NFAT5Δ-tdTomato and FLAG-HES1 in HEK293A cells. h A schematic model showing the prediction of HES1 binding motifs in the BGT1 promoter using JASPAR. The predicted sites of HES1 binding are presented as white boxes with x-markers. ( i ) ChIP analysis evaluating HES1 recruitment to a predicted site (HES1-c site) in the BGT1 promoter under high salt conditions at 5.5 h after ISO and high NaCl stimuli, 400 mOsm. j A schematic model showing the structure of the BGT1 reporter and mutations of the HES1 binding site introduced into the reporter. k Effect of HES1 depletion on BGT1 reporter activity at 9 h after ISO and high NaCl stimuli, 400 mOsm ( n = 3). l Effect of a double point mutant (Mutant) and deletion mutant (Deletion) in the HES1 binding site on the BGT1 reporter activity ( n = 4). Mutant sequences shown in ( j ).In the bar graphs, individual values (white points) and the mean ± SEM are presented. * p < 0.05, ** p < 0.01, *** p < 0.001. ISO, 300 mOsm; high NaCl, 400 mOsm. See also Supplementary Fig. .

Article Snippet: The following antibodies were used for immunoblots(IB) or immunoprecipitation(IP) or immunofluorescence(IF) or Chromatin-IP(ChIP): FLAG antibody (1E6) (IB, Wako Pure Chemicals Industries, #014-22383); NFAT5 antibody (IB, IF, Santa Cruz Biotechnology, sc-13035; ChIP, Santa Cruz Biotechnology, sc-398171); HES1 antibody (IB, ChIP, Abcam, ab196328); LAMIN A/C antibody (IB, Cell Signaling Technology, #4777); beta-tubulin antibody (IB, Santa Cruz Biotechnology, sc-5274); alpha-tubulin antibody (IB, Santa Cruz Biotechnology, sc-53029); p44/42 MAPK (ERK1/2) antibody (IB, Cell Signaling Technology, #9102); phospho-p44/42 MAPK (ERK1/2) antibody (IB, Cell Signaling Technology, #9106); c-Myc antibody (IB, Sigma-Aldrich, A3853); Notch1 antibody (IB, Cell Signaling Technology, #4380); Cleaved Notch1 (Val1744) antibody (IB, Cell Signaling Technology, #4147); MEK1/2 antibody (IB, Cell Signaling Technology, #9122); DsRed antibody (IB, Santa Cruz Biotechnology, sc-101526); Anti-rebbit IgG, HRP-linked Antibody (IB, Cell Signaling Technology, #7074); Anti-mouse IgG, HRP-linked Antibody (IB, Cell Signaling Technology, #7076); Anti-rat IgG, HRP-linked Antibody (IB, Cell Signaling Technology, #7077).

Techniques: Binding Assay, Co-Immunoprecipitation Assay, Activity Assay, Mutagenesis

Journal: Communications Biology

Article Title: HES1 potentiates high salt stress response as an enhancer of NFAT5-DNA binding

doi: 10.1038/s42003-024-06997-7

Figure Lengend Snippet: HES1 potentiates DNA binding and nuclear translocation of NFAT5 as a positive regulator. Mechanistically, HES1 is induced by high salinity/hyperosmotic stress via ERK signaling and facilitates NFAT5 recruitment to its target promoter region through protein binding to NFAT5 and DNA binding to adjacent site of NFAT5 binding sites. The regulation is essential for the proper induction of osmoprotective genes and cytoprotection under high salt stress. Hes1 induced through the canonical Notch signaling can concomitantly contribute to NFAT5 nuclear translocation when it is activated.

Article Snippet: The following antibodies were used for immunoblots(IB) or immunoprecipitation(IP) or immunofluorescence(IF) or Chromatin-IP(ChIP): FLAG antibody (1E6) (IB, Wako Pure Chemicals Industries, #014-22383); NFAT5 antibody (IB, IF, Santa Cruz Biotechnology, sc-13035; ChIP, Santa Cruz Biotechnology, sc-398171); HES1 antibody (IB, ChIP, Abcam, ab196328); LAMIN A/C antibody (IB, Cell Signaling Technology, #4777); beta-tubulin antibody (IB, Santa Cruz Biotechnology, sc-5274); alpha-tubulin antibody (IB, Santa Cruz Biotechnology, sc-53029); p44/42 MAPK (ERK1/2) antibody (IB, Cell Signaling Technology, #9102); phospho-p44/42 MAPK (ERK1/2) antibody (IB, Cell Signaling Technology, #9106); c-Myc antibody (IB, Sigma-Aldrich, A3853); Notch1 antibody (IB, Cell Signaling Technology, #4380); Cleaved Notch1 (Val1744) antibody (IB, Cell Signaling Technology, #4147); MEK1/2 antibody (IB, Cell Signaling Technology, #9122); DsRed antibody (IB, Santa Cruz Biotechnology, sc-101526); Anti-rebbit IgG, HRP-linked Antibody (IB, Cell Signaling Technology, #7074); Anti-mouse IgG, HRP-linked Antibody (IB, Cell Signaling Technology, #7076); Anti-rat IgG, HRP-linked Antibody (IB, Cell Signaling Technology, #7077).

Techniques: Binding Assay, Translocation Assay, Protein Binding

Journal: Nature Immunology

Article Title: Sodium chloride in the tumor microenvironment enhances T cell metabolic fitness and cytotoxicity

doi: 10.1038/s41590-024-01918-6

Figure Lengend Snippet: a , Real-time killing assay with nucleofected MART-1-specific T cells and A375 melanoma cell target cells at a 1:1 ratio under high and low NaCl conditions using the xCELLigence technology. Left, the normalized cell index; middle, the specific lysis; right, the cumulative quantification of 3T cell donors ( n = 3 experiments; mean ± s.e.m.; two-way ANOVA, * P < 0.05). b , Murine ROR1 CAR T cells generated and cultured for 48 h under high and low NaCl conditions and then cocultured with ROR1-expressing target cells at a 10:1 ratio. Antigen-specific lysis of Panc02-ROR1 cells by CD8 + CAR T cells was determined at different time points ( n = 3 independent experiments; mean ± s.d., two-way ANOVA). c , Experimental design. d , The tumor growth curves of subcutaneous tumors. Tumor growth was normalized to the tumor size on the day of CD8 + T cell injection ( n = 7 (PancOVA), n = 6 (PancOVA + low NaCl control (CTL)), n = 6 (PancOVA + high NaCl CTL); mean ± s.e.m. two-way ANOVA with Tukey’s honestly significant difference (HSD), multiple-comparison test). e , f , Flow cytometric analysis of intratumoral CD45.2 + CD8 + T cells 72 h after T cell transfer ( n = 6 ( e ), n = 5 ( f ); mean ± s.d., two-tailed, unpaired Student’s t -test). g , ScRNA-seq and module score calculation for T cell cytotoxicity genes obtained from published reports , , validated with genes from GO:0001916 ( P = 0.01). Intratumoral CD8 + T cells are shown from 56 patients with pancreatic cancer (from accession nos. GSE155698 , GSE111672 , GSE154778 , GSM4293555 and PRJCA001063 ) , integration of all cells: 10.5281/zenodo.6024273. CD8 + T cells were categorized into cells with a high and low NaCl signature based on the NaCl signature obtained from scRNA-seq of CD8 + CD45RA – T cells treated under high versus low NaCl concentrations (top 60 upregulated DEGs; Supplementary Table ; cutoff defined as module score ≥0 and <0 for high versus low NaCl signature, respectively; Wilcoxon’s rank-sum test). h , i , Kaplan–Meier tumor-free survival probability of patients from TCGA database diagnosed with pancreatic cancer. Patients were subgrouped by computing an optimal cutoff for NFAT5 ( h ) and ATP1A1 ( i ) expression. TPM values were normalized toward overall survival outcome. Number of patient samples: pancreatic cancer: n = 72 for NFAT5 high, n = 9 for NFAT5 low; n = 41 for ATP1A1 high; n = 40 for ATP2A2 low; significance of survival differences was determined using the Peto–Peto algorithm with the surv_pvalue function (method = ‘S1’) as implemented in the R package survminer.

Article Snippet: The following antibodies were used: mouse anti-human NFAT5 antibody (Santa Cruz, cat. no. sc-398171), rabbit anti-human Lamin-B1 antibody (Cell Signaling Technology, cat. no. D4Q4Z), mouse anti-human β-actin (Cell Signaling Technology, cat. no. 8H10D10) antibody.

Techniques: Lysis, Generated, Cell Culture, Expressing, Injection, Control, Comparison, Two Tailed Test

Journal: Biological Research

Article Title: Placental growth factor mediates pathological uterine angiogenesis by activating the NFAT5-SGK1 signaling axis in the endometrium: implications for preeclampsia development

doi: 10.1186/s40659-024-00526-w

Figure Lengend Snippet: PlGF activates NFAT5 expression and activity in EnSCs. ( a ) NFAT5 mRNA transcript kinetics in EnSCs treated with PlGF for 2, 4 and 6 days at a concentration of 20 ng/ml. L19 was used as a housekeeping gene and the data was normalized to untreated (Con) ( n = 5, **, p < 0.01). ( b ) Original Western blot analysis of NFAT5 protein with GAPDH as loading control in untreated (Con) and PlGF treated EnSCs. ( c ) Average NFAT5 protein levels after 6 days treatment with PlGF ( n = 5, ** p < 0.01). The samples are represented after normalization with untreated control (Con). d ) Immunofluorescence images confirms nuclear translocation of NFAT5 from the cytoplasm when activated by PlGF ( n = 3). Scale bar: 20 μm. Data represented as arithmetic mean ± SEM. Significance was determined using student’s unpaired two-tailed t-test with Welch’s correction method. n represents the number of independent experiments (biological replicates)

Article Snippet: After incubation with 5% non-fat milk or BSA in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20) for 60 min, the membrane was washed once with TBST and incubated with primary antibodies against NFAT5 (1:2000, #NB20-3446, Novus Biologicals) [ ], SGK1 (1:1000, #07-315, Merck) [ ], phospho-SGK1 (1:1000, #36 − 002, Merck) [ ], p38 MAPK (1:1000, #8690S, Cell Signaling Technologies) [ ], phospho-p38 MAPK (1:1000, #4511, Cell Signaling Technologies) [ ], VEGF-A (1:3000, #ab46154, abcam) [ ], VEGFR1 (1:1000, #2893, Cell Signaling Technologies) [ ], VEGFR2 (1:1000, #2479, Cell Signaling Technologies) [ ], or GAPDH (1:1000, #5174, Cell Signaling Technologies) [ ] at 4 °C for overnight.

Techniques: Expressing, Activity Assay, Concentration Assay, Western Blot, Control, Immunofluorescence, Translocation Assay, Two Tailed Test

Journal: Biological Research

Article Title: Placental growth factor mediates pathological uterine angiogenesis by activating the NFAT5-SGK1 signaling axis in the endometrium: implications for preeclampsia development

doi: 10.1186/s40659-024-00526-w

Figure Lengend Snippet: PlGF-NFAT5 angiogenic signaling axis in EnSCs. ( a ) Original Western blots of total and phosphorylated levels of p38 MAPK, SGK1 and total VEGF-A targets with GAPDH as loading control in untreated (Con)and PlGF treated EnSCs. ( b - e ) Average protein expression levels of total and phosphorylated levels of p38 MAPK and SGK1 targets in untreated (Con) and PlGF treated EnSCs ( n = 5, *, p < 0.05). ( f ) qPCR analysis of HIF-1α transcript levels in untreated (Con) and PlGF treated EnSCs. L19 was used as a housekeeping gene ( n = 5, *, p < 0.05). ( g ) Luciferase reporter assay measuring the HIF-1α promoter activity in untreated (Con), PlGF and DMOG (positive control for hypoxia, 0.5 mM for 24 h) treated EnSCs ( n = 5, ***, p < 0.001, ****, p < 0.0001). ( h ) Immunoblotting showing average protein expression levels of VEGF-A (Con) in untreated and PlGF treated EnSCs ( n = 5, **, p < 0.01). ( i ) Supernatant from untreated (Con) and PlGF treated EnSCs was collected and secreted VEGF-A levels were quantified with ELISA ( n = 5, *, p < 0.05). Data represented here as arithmetic mean ± SEM. The treatment samples groups (PlGF) are represented after normalization with untreated control (Con). Significance was determined using student’s unpaired two-tailed t-test with Welch’s correction method. n represents the number of independent experiments (biological replicates)

Article Snippet: After incubation with 5% non-fat milk or BSA in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20) for 60 min, the membrane was washed once with TBST and incubated with primary antibodies against NFAT5 (1:2000, #NB20-3446, Novus Biologicals) [ ], SGK1 (1:1000, #07-315, Merck) [ ], phospho-SGK1 (1:1000, #36 − 002, Merck) [ ], p38 MAPK (1:1000, #8690S, Cell Signaling Technologies) [ ], phospho-p38 MAPK (1:1000, #4511, Cell Signaling Technologies) [ ], VEGF-A (1:3000, #ab46154, abcam) [ ], VEGFR1 (1:1000, #2893, Cell Signaling Technologies) [ ], VEGFR2 (1:1000, #2479, Cell Signaling Technologies) [ ], or GAPDH (1:1000, #5174, Cell Signaling Technologies) [ ] at 4 °C for overnight.

Techniques: Western Blot, Control, Expressing, Luciferase, Reporter Assay, Activity Assay, Positive Control, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Biological Research

Article Title: Placental growth factor mediates pathological uterine angiogenesis by activating the NFAT5-SGK1 signaling axis in the endometrium: implications for preeclampsia development

doi: 10.1186/s40659-024-00526-w

Figure Lengend Snippet: Angiogenic effect of PlGF-NFAT5 signaling axis on HUVECs. ( a ) Schematics describing the experimental approach of CM treatment on HUVECs. ( b ) BrdU incorporated ELISA analysis for cell proliferation measured in Con-CM and PlGF-CM treated HUVECs ( n = 4, *, p < 0.05). ( c ) Representative fluorescence microscopic images of wound healing scratch assay on Con-CM and PlGF-CM treated HUVECs at 0 and 24 h ( n = 4). Yellow line represents the wound area created. Scale bar: 650 μm. ( d ) Wound closure rate in Con-CM and PlGF-CM treated HUVECs at 24 h ( n = 4, **, p < 0.01) explain normalization. ( e ) Representative fluorescence microscopic images of tube formation assay on a matrigel with Con-CM, PlGF-CM and DMOG (positive control; 0.5 mM for 24 h) treated HUVECs at 24 h ( n = 4). The insert displays HUVECs seeded on the matrigel at 0 h. Scale bar: 650 μm. ( f ) Tube formation assay analysis showing tube length in Con-CM, PlGF-CM and DMOG treated HUVECs at 24 h ( n = 4). ( g ) Tube formation assay analysis depicting number of branches in Con-CM, PlGF-CM and DMOG treated HUVECs at 24 h ( n = 4, *, p < 0.05). ( h - l ) qPCR analysis of Notch receptors ( Notch 1 and Notch 2 ), ligands ( Dll4 and Jagged-1 ) and target genes ( Hey 1 ) in Con-CM and PlGF-CM treated HUVECs. L19 was used as a housekeeping control. ( n = 4, *, p < 0.05, **, p < 0.01). ( m ) Original Western blots of VEGFR1, VEGFR2 and VEGF-A targets with GAPDH as loading control in Con-CM and PlGF-CM treated HUVECs. ( n ) Average protein levels of VEGFR1, VEGFR2 and VEGF-A in Con-CM and PlGF-CM treated HUVECs ( n = 4, *, p < 0.05, **, p < 0.01). Data represented here as arithmetic mean ± SEM. The treatment samples groups (PlGF-CM) are represented after normalization with control (Con-CM). Significance was determined using student’s unpaired two-tailed t-test with Welch’s correction method. ( o ) EIS analysis of cell impedance values in Con-CM and PlGF-CM treated HUVEC monolayer representing endothelial barrier function ( n = 4, ****, p < 0.0001). Significance was determined using student’s unpaired two-tailed t-test with Welch’s correction method for cell impedance values at 4 h. n represents the number of independent experiments (biological replicates)

Article Snippet: After incubation with 5% non-fat milk or BSA in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20) for 60 min, the membrane was washed once with TBST and incubated with primary antibodies against NFAT5 (1:2000, #NB20-3446, Novus Biologicals) [ ], SGK1 (1:1000, #07-315, Merck) [ ], phospho-SGK1 (1:1000, #36 − 002, Merck) [ ], p38 MAPK (1:1000, #8690S, Cell Signaling Technologies) [ ], phospho-p38 MAPK (1:1000, #4511, Cell Signaling Technologies) [ ], VEGF-A (1:3000, #ab46154, abcam) [ ], VEGFR1 (1:1000, #2893, Cell Signaling Technologies) [ ], VEGFR2 (1:1000, #2479, Cell Signaling Technologies) [ ], or GAPDH (1:1000, #5174, Cell Signaling Technologies) [ ] at 4 °C for overnight.

Techniques: Enzyme-linked Immunosorbent Assay, Fluorescence, Wound Healing Assay, Tube Formation Assay, Positive Control, Control, Western Blot, Two Tailed Test

Journal: Biological Research

Article Title: Placental growth factor mediates pathological uterine angiogenesis by activating the NFAT5-SGK1 signaling axis in the endometrium: implications for preeclampsia development

doi: 10.1186/s40659-024-00526-w

Figure Lengend Snippet: Graphical abstract describing the effect of pathological PlGF levels in altered uterine endometrial angiogenesis and its plausible role in PE pathology. Aberrant levels of endometrial PlGF activates NFAT5-SGK1-VEGF-A signaling axis in uterine stromal cells. Activation of this signaling cascade presents negative angiogenic cues to endothelial cells, with deregulated secreted protein cargo (decreased angiogenic factor VEGF-A and increased ECM associated proteins). PlGF mediated secreted factors supports abnormal vessel development in HUVECs, with dysregulation of Notch-VEGF signaling. Aberrant PlGF triggered stromal-endothelial paracrine signaling results in hypersprouting, high cellular resistance and impaired BeWo invasion through HUEVCs. Hypersprouting and high cellular impedance in HUVECs confirm pathological uterine vascularization upon deregulated endometrial PlGF. Thus, we postulate such aberrant uterine angiogenesis prior to pregnancy will likely lead to poor quality maternal vessels, inadequate trophoblast invasion causing poor placentation as seen in PE pregnancy (Images created with BioRender)

Article Snippet: After incubation with 5% non-fat milk or BSA in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween 20) for 60 min, the membrane was washed once with TBST and incubated with primary antibodies against NFAT5 (1:2000, #NB20-3446, Novus Biologicals) [ ], SGK1 (1:1000, #07-315, Merck) [ ], phospho-SGK1 (1:1000, #36 − 002, Merck) [ ], p38 MAPK (1:1000, #8690S, Cell Signaling Technologies) [ ], phospho-p38 MAPK (1:1000, #4511, Cell Signaling Technologies) [ ], VEGF-A (1:3000, #ab46154, abcam) [ ], VEGFR1 (1:1000, #2893, Cell Signaling Technologies) [ ], VEGFR2 (1:1000, #2479, Cell Signaling Technologies) [ ], or GAPDH (1:1000, #5174, Cell Signaling Technologies) [ ] at 4 °C for overnight.

Techniques: Activation Assay

Journal: Biological Research

Article Title: Placental growth factor mediates pathological uterine angiogenesis by activating the NFAT5-SGK1 signaling axis in the endometrium: implications for preeclampsia development

doi: 10.1186/s40659-024-00526-w

Figure Lengend Snippet: PlGF activates NFAT5 expression and activity in EnSCs. ( a ) NFAT5 mRNA transcript kinetics in EnSCs treated with PlGF for 2, 4 and 6 days at a concentration of 20 ng/ml. L19 was used as a housekeeping gene and the data was normalized to untreated (Con) ( n = 5, **, p < 0.01). ( b ) Original Western blot analysis of NFAT5 protein with GAPDH as loading control in untreated (Con) and PlGF treated EnSCs. ( c ) Average NFAT5 protein levels after 6 days treatment with PlGF ( n = 5, ** p < 0.01). The samples are represented after normalization with untreated control (Con). d ) Immunofluorescence images confirms nuclear translocation of NFAT5 from the cytoplasm when activated by PlGF ( n = 3). Scale bar: 20 μm. Data represented as arithmetic mean ± SEM. Significance was determined using student’s unpaired two-tailed t-test with Welch’s correction method. n represents the number of independent experiments (biological replicates)

Article Snippet: The slides were then incubated with primary antibodies for NFAT5 (1:200, #NB20-3446, Novus Biologicals) [ ] at 4 °C overnight.

Techniques: Expressing, Activity Assay, Concentration Assay, Western Blot, Control, Immunofluorescence, Translocation Assay, Two Tailed Test

Journal: Biological Research

Article Title: Placental growth factor mediates pathological uterine angiogenesis by activating the NFAT5-SGK1 signaling axis in the endometrium: implications for preeclampsia development

doi: 10.1186/s40659-024-00526-w

Figure Lengend Snippet: PlGF-NFAT5 angiogenic signaling axis in EnSCs. ( a ) Original Western blots of total and phosphorylated levels of p38 MAPK, SGK1 and total VEGF-A targets with GAPDH as loading control in untreated (Con)and PlGF treated EnSCs. ( b - e ) Average protein expression levels of total and phosphorylated levels of p38 MAPK and SGK1 targets in untreated (Con) and PlGF treated EnSCs ( n = 5, *, p < 0.05). ( f ) qPCR analysis of HIF-1α transcript levels in untreated (Con) and PlGF treated EnSCs. L19 was used as a housekeeping gene ( n = 5, *, p < 0.05). ( g ) Luciferase reporter assay measuring the HIF-1α promoter activity in untreated (Con), PlGF and DMOG (positive control for hypoxia, 0.5 mM for 24 h) treated EnSCs ( n = 5, ***, p < 0.001, ****, p < 0.0001). ( h ) Immunoblotting showing average protein expression levels of VEGF-A (Con) in untreated and PlGF treated EnSCs ( n = 5, **, p < 0.01). ( i ) Supernatant from untreated (Con) and PlGF treated EnSCs was collected and secreted VEGF-A levels were quantified with ELISA ( n = 5, *, p < 0.05). Data represented here as arithmetic mean ± SEM. The treatment samples groups (PlGF) are represented after normalization with untreated control (Con). Significance was determined using student’s unpaired two-tailed t-test with Welch’s correction method. n represents the number of independent experiments (biological replicates)

Article Snippet: The slides were then incubated with primary antibodies for NFAT5 (1:200, #NB20-3446, Novus Biologicals) [ ] at 4 °C overnight.

Techniques: Western Blot, Control, Expressing, Luciferase, Reporter Assay, Activity Assay, Positive Control, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Biological Research

Article Title: Placental growth factor mediates pathological uterine angiogenesis by activating the NFAT5-SGK1 signaling axis in the endometrium: implications for preeclampsia development

doi: 10.1186/s40659-024-00526-w

Figure Lengend Snippet: Angiogenic effect of PlGF-NFAT5 signaling axis on HUVECs. ( a ) Schematics describing the experimental approach of CM treatment on HUVECs. ( b ) BrdU incorporated ELISA analysis for cell proliferation measured in Con-CM and PlGF-CM treated HUVECs ( n = 4, *, p < 0.05). ( c ) Representative fluorescence microscopic images of wound healing scratch assay on Con-CM and PlGF-CM treated HUVECs at 0 and 24 h ( n = 4). Yellow line represents the wound area created. Scale bar: 650 μm. ( d ) Wound closure rate in Con-CM and PlGF-CM treated HUVECs at 24 h ( n = 4, **, p < 0.01) explain normalization. ( e ) Representative fluorescence microscopic images of tube formation assay on a matrigel with Con-CM, PlGF-CM and DMOG (positive control; 0.5 mM for 24 h) treated HUVECs at 24 h ( n = 4). The insert displays HUVECs seeded on the matrigel at 0 h. Scale bar: 650 μm. ( f ) Tube formation assay analysis showing tube length in Con-CM, PlGF-CM and DMOG treated HUVECs at 24 h ( n = 4). ( g ) Tube formation assay analysis depicting number of branches in Con-CM, PlGF-CM and DMOG treated HUVECs at 24 h ( n = 4, *, p < 0.05). ( h - l ) qPCR analysis of Notch receptors ( Notch 1 and Notch 2 ), ligands ( Dll4 and Jagged-1 ) and target genes ( Hey 1 ) in Con-CM and PlGF-CM treated HUVECs. L19 was used as a housekeeping control. ( n = 4, *, p < 0.05, **, p < 0.01). ( m ) Original Western blots of VEGFR1, VEGFR2 and VEGF-A targets with GAPDH as loading control in Con-CM and PlGF-CM treated HUVECs. ( n ) Average protein levels of VEGFR1, VEGFR2 and VEGF-A in Con-CM and PlGF-CM treated HUVECs ( n = 4, *, p < 0.05, **, p < 0.01). Data represented here as arithmetic mean ± SEM. The treatment samples groups (PlGF-CM) are represented after normalization with control (Con-CM). Significance was determined using student’s unpaired two-tailed t-test with Welch’s correction method. ( o ) EIS analysis of cell impedance values in Con-CM and PlGF-CM treated HUVEC monolayer representing endothelial barrier function ( n = 4, ****, p < 0.0001). Significance was determined using student’s unpaired two-tailed t-test with Welch’s correction method for cell impedance values at 4 h. n represents the number of independent experiments (biological replicates)

Article Snippet: The slides were then incubated with primary antibodies for NFAT5 (1:200, #NB20-3446, Novus Biologicals) [ ] at 4 °C overnight.

Techniques: Enzyme-linked Immunosorbent Assay, Fluorescence, Wound Healing Assay, Tube Formation Assay, Positive Control, Control, Western Blot, Two Tailed Test

Journal: Biological Research

Article Title: Placental growth factor mediates pathological uterine angiogenesis by activating the NFAT5-SGK1 signaling axis in the endometrium: implications for preeclampsia development

doi: 10.1186/s40659-024-00526-w

Figure Lengend Snippet: Graphical abstract describing the effect of pathological PlGF levels in altered uterine endometrial angiogenesis and its plausible role in PE pathology. Aberrant levels of endometrial PlGF activates NFAT5-SGK1-VEGF-A signaling axis in uterine stromal cells. Activation of this signaling cascade presents negative angiogenic cues to endothelial cells, with deregulated secreted protein cargo (decreased angiogenic factor VEGF-A and increased ECM associated proteins). PlGF mediated secreted factors supports abnormal vessel development in HUVECs, with dysregulation of Notch-VEGF signaling. Aberrant PlGF triggered stromal-endothelial paracrine signaling results in hypersprouting, high cellular resistance and impaired BeWo invasion through HUEVCs. Hypersprouting and high cellular impedance in HUVECs confirm pathological uterine vascularization upon deregulated endometrial PlGF. Thus, we postulate such aberrant uterine angiogenesis prior to pregnancy will likely lead to poor quality maternal vessels, inadequate trophoblast invasion causing poor placentation as seen in PE pregnancy (Images created with BioRender)

Article Snippet: The slides were then incubated with primary antibodies for NFAT5 (1:200, #NB20-3446, Novus Biologicals) [ ] at 4 °C overnight.

Techniques: Activation Assay