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: As a control, the constitutive transcriptional activator VP16 was transfected using pEGFP-TetR-NLS-VP16 (Addgene plasmid #103834).
Techniques: Fluorescence, Control, Reporter Assay, Activation Assay
Addgene inc is a verified supplier 
![( 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 <t>VP16</t> 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 <t>transcriptional</t> 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.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_8016/pmc11838016/pmc11838016__sciadv.adu3194-f8.jpg)