nfat5  (TargetMol)

Journal: Communications Biology

Article Title: Wnt11 mediates fibroblast–smooth muscle cell interaction to promote neurogenic bladder fibrosis in rats

doi: 10.1038/s42003-026-09647-2

Figure Lengend Snippet: Relative expression levels of Vangl2 and p-Smad2/3 in BFs ( a ) and SMCs ( b ) treated with TGF-β1 with or without siVangl2 by western blotting ( n = 3). Interaction between endogenous TβR1 and Vangl2 in BFs ( c ) and SMCs ( d ). e Confocal microscopy analysis of BFs and SMCs showing the colocalization of Vangl2 (green) and TβR1 (red). Scale bar = 20 μm. Quantification of colocalization was determined by ImageJ software in five randomly chosen fields. Numbers in the images correspond to the average Pearson’s correlation coefficient ± SD. Representative western blotting and quantitative analyses for the phosphorylation levels of JNK and c-JUN in BFs ( f ) and SMCs ( g ). ( n = 3). h Heat map showing differentially expressed genes in the NT signaling pathway based on RNA-seq data in TGF-β1-stimulated BFs with or without siVangl2 ( n = 3). i Representative western blotting analyses for total and intranuclear NFAT5 in BFs treated with SP600125. j Representative western blotting showing KRN5 inhibiting the phosphorylation level of Smad2/3 in BFs with TGF-β1 treatment. Immunoprecipitation demonstrated that NFAT5 bound to JNK ( k ) and Smad2/3 ( l ) in BFs. Data are shown as the mean ± SD. P values were determined by the two-way ANOVA followed by Tukey’s post-hoc test ( a , b , f , g ), * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: For pharmacologic inhibition of TGF-β1-dependent pathways and WNT11-mediated planar cell polarity (PCP) pathway, cells were incubated with JNK inhibitor SP600125 (20 μM, #HY-12041, MedChemExpress), NFAT5 inhibitor KRN5 (1 μM, #HY-112126, MedChemExpress), p38 inhibitor SB202190 (10 μM, #HY-10295, MedChemExpress), Rock inhibitor Y27632 (5 μM, #HY-10071, MedChemExpress), Rac inhibitor NSC23766 (200 μM, #HY-15723, MedChemExpress), Smad2/3 inhibitor TP0427736 (20 nM, #HY-118528, MedChemExpress), and TβR1 inhibitor PF06952229 (10 μM, #HY-136244, MedChemExpress) for 24 h after incubation of serum-free medium for 24 h.

Techniques: Expressing, Western Blot, Confocal Microscopy, Software, Phospho-proteomics, RNA Sequencing, Immunoprecipitation

Journal: Genes & Diseases

Article Title: Microglial NFAT5 aggravates neuroinflammation via mediating NLRP6 inflammasome in experimental ischemic stroke

doi: 10.1016/j.gendis.2025.101614

Figure Lengend Snippet: Increased expression of NFAT5 in microglia after OGD/R and MCAO modeling. (A) Schematic of three cell lines undergoing OGD/R modeling. BV2, mouse microglia cell line; MA, mouse astrocyte cell line; HT22, mouse hippocampal neuron cell line. (B) Western blots for NFAT5 in BV2, MA, and HT22 cell lines after OGD/R modeling ( n = 3). (C) The NFAT5 protein level in the nuclei and cytoplasm of BV2 cells was detected by western blotting ( n = 3). (D) Representative immunofluorescence images of NFAT5 in BV2 cells (bar = 25 μm). (E) The overlap coefficient of DAPI and NFAT5 in BV2 cells ( n = 3). (F, G) Immunofluorescence for NFAT5 in microglia from peri-infarct brain tissues of mice (bar = 50 μm). Iba-1 was marked in green for microglia, and NFAT5 was marked in red. The cells indicated by the white arrows were microglia. Scale bar in magnified view: 10 μm. The data were presented as mean with standard deviation. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗∗ p < 0.0001.

Article Snippet: Next, we obtained BV2 cells with stable NFAT5 knockdown by 2 μg/mL puromycin (MCE, USA) treatment.

Techniques: Expressing, Western Blot, Immunofluorescence, Standard Deviation

Journal: Genes & Diseases

Article Title: Microglial NFAT5 aggravates neuroinflammation via mediating NLRP6 inflammasome in experimental ischemic stroke

doi: 10.1016/j.gendis.2025.101614

Figure Lengend Snippet: Rescue of MCAO-induced cerebral infarction and neurological deficits in mice through microglial NFAT5 interference. (A) Design of adeno-associated virus (AAV) to knock down microglial NFAT5. (B, C) Representative immunofluorescence images depicting Iba-1 (in pink) and NFAT5 (in red) (B) and quantification of NFAT5 fluorescence intensity in Iba-1 positive cells (C). The AAV expressed enhanced green fluorescent protein (EGFP). The infected microglia were delineated with circular and square annotations. The magnified view focused on the microglia within the square annotation. Scale bar: 50 μm. Scale bar in magnified view: 5 μm. The data were presented as mean with standard deviation ( n = 3). (D, G) Representative images and quantification of magnetic resonance imaging data. The data were presented as median ( n = 5). (E, F) Maximum and mean mouse limb grip strength ( n = 12). The data were presented as mean with standard deviation. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: Next, we obtained BV2 cells with stable NFAT5 knockdown by 2 μg/mL puromycin (MCE, USA) treatment.

Techniques: Virus, Knockdown, Immunofluorescence, Fluorescence, Infection, Standard Deviation, Magnetic Resonance Imaging

Journal: Genes & Diseases

Article Title: Microglial NFAT5 aggravates neuroinflammation via mediating NLRP6 inflammasome in experimental ischemic stroke

doi: 10.1016/j.gendis.2025.101614

Figure Lengend Snippet: Microglial NFAT5 knockdown mitigates MCAO-induced brain morphological damage and apoptosis. (A) Representative images of hematoxylin-eosin staining in the hippocampus and cortex of mice. Scale bar: 50 μm ( n = 3). (B – D) Representative images of Nissel staining (D) and quantification of Nissel-positive cells in the hippocampus (B) and cortex (C). Scale bar: 50 μm. The data were presented as mean with standard deviation ( n = 3). (E, F) Representative TUNEL assay images in cortical brain tissue regions (F) and quantification of TUNEL-positive cells per 0.1 mm 2 (E). TUNEL staining is shown in green, and nuclei are labeled in blue ( n = 3). Scale bar: 50 μm. (G – I) Western blotting analysis of Bcl-2 and Bax expression levels ( n = 3). Data presented as means with standard deviation. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001.

Article Snippet: Next, we obtained BV2 cells with stable NFAT5 knockdown by 2 μg/mL puromycin (MCE, USA) treatment.

Techniques: Knockdown, Staining, Standard Deviation, TUNEL Assay, Labeling, Western Blot, Expressing

Journal: Genes & Diseases

Article Title: Microglial NFAT5 aggravates neuroinflammation via mediating NLRP6 inflammasome in experimental ischemic stroke

doi: 10.1016/j.gendis.2025.101614

Figure Lengend Snippet: Microglial NFAT5 silencing attenuates neuronal apoptosis in OGD/R model. (A) Schematic representation of BV2 (mouse microglia cell line) conditioned medium treatment on HT22 (mouse hippocampal neuron cell line). (B) A lactate dehydrogenase (LDH) assay was used to detect the released LDH in HT22 medium ( n = 8–12). ( C ) CCK-8 assay was used to measure the cell survival rate of HT22 ( n = 9). (D, E) Representative images and quantification of flow cytometry illustrating the percentage of annexin V-FITC and propidium iodide (PI)-labeled HT22 cells ( n = 3). (F–I) Representative immunofluorescence images of Bcl-2 (F) and Bax (H) and quantification of fluorescence intensity for Bcl-2 (G) and Bax (I) in HT22 cells ( n = 3). Scale bar: 25 μm. The data were presented as mean with standard deviation. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001.

Article Snippet: Next, we obtained BV2 cells with stable NFAT5 knockdown by 2 μg/mL puromycin (MCE, USA) treatment.

Techniques: Lactate Dehydrogenase Assay, CCK-8 Assay, Flow Cytometry, Labeling, Immunofluorescence, Fluorescence, Standard Deviation

Journal: Genes & Diseases

Article Title: Microglial NFAT5 aggravates neuroinflammation via mediating NLRP6 inflammasome in experimental ischemic stroke

doi: 10.1016/j.gendis.2025.101614

Figure Lengend Snippet: NFAT5 inhibition ameliorates microglia-mediated neuroinflammation and NLRP6 inflammasome activation in MCAO model. (A – E) Western blotting analysis of pro-inflammatory factor protein levels (IL-1β, TNF-α, and IL-6) in brain tissues (A), with protein levels normalized to the sham group (B–E) ( n = 3). (F, G) Representative immunofluorescence images of Iba-1 (F) and quantification of Iba-1-positive cells (G) in brain sections ( n = 3). Scale bar: 50 μm. (H, I) Representative immunofluorescence images of MPO (H) and quantification of MPO-positive cells (I) in brain sections ( n = 3). Scale bar: 40 μm. (J – N) Western blotting analysis was applied for NLRP6, ASC, pro-caspase-1, and cleaved-caspase-1 in brain tissue and the protein levels were normalized to the sham group ( n = 3). (O) Quantification of microglial morphology ( n = 3). The data were presented as mean with standard deviation. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001; ns, no statistical significance.

Article Snippet: Next, we obtained BV2 cells with stable NFAT5 knockdown by 2 μg/mL puromycin (MCE, USA) treatment.

Techniques: Inhibition, Activation Assay, Western Blot, Immunofluorescence, Standard Deviation

Journal: Genes & Diseases

Article Title: Microglial NFAT5 aggravates neuroinflammation via mediating NLRP6 inflammasome in experimental ischemic stroke

doi: 10.1016/j.gendis.2025.101614

Figure Lengend Snippet: NFAT5 silencing suppresses inflammatory response and NLRP6 inflammasome activation in OGD/R Model. (A – F) Western blotting analysis of NFAT5, pro-IL-1β, IL-1β, TNF-α, and IL-6 protein levels in BV2 cells (A), with the protein levels normalized to the sh-NC control group (B–F) ( n = 3). (G – I) ELISA measurement of IL-1β, TNF-α, and IL-6 concentrations in BV2 cell culture medium ( n = 3). (K–N) Western blotting analysis was performed for NLRP6, pro-caspase-1, and cleaved-caspase-1 in BV2 cells, with the protein levels normalized to the sh-NC control group ( n = 3). (J) The Nlrp6 mRNA level in BV2 cells was detected by quantitative PCR ( n = 3). The data were presented as mean with standard deviation. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001; ns, no statistical significance.

Article Snippet: Next, we obtained BV2 cells with stable NFAT5 knockdown by 2 μg/mL puromycin (MCE, USA) treatment.

Techniques: Activation Assay, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Cell Culture, Real-time Polymerase Chain Reaction, Standard Deviation

Journal: Genes & Diseases

Article Title: Microglial NFAT5 aggravates neuroinflammation via mediating NLRP6 inflammasome in experimental ischemic stroke

doi: 10.1016/j.gendis.2025.101614

Figure Lengend Snippet: NFAT5 is a transcription factor for the Nlrp6 promoter. (A) The diagram illustrating the predicted binding sites of NFAT5 on the Nlrp6 promoter. (B, C) Dual luciferase reporter assay of pGL4.10- Nlrp6 promoter after co-transfection with NFAT5 overexpressing (NFAT5) or control vector (TR) in N2A and 293T cell lines ( n = 8). (D) Construction of two Nlrp6 promoter fragments (P1 and P2) and a mutant of the Nlrp6 promoter. (E) Dual luciferase reporter assay of pGL4.10- Nlrp6 promoter (full length) or pGL4.10- Nlrp6 promoter fragments after co-transfection with NFAT5 overexpressing (NFAT5) or control vector (TR) into 293T cell line ( n = 4). (F) Dual luciferase reporter assay of pGL4.10- Nlrp6 promoter (full length) or pGL4.10- Nlrp6 promoter mutant after co-transfection with NFAT5-overexpressing (NFAT5) or control vector (TR) into 293T cell line ( n = 4). (G) The schematic depicting the positions of the Nlrp6 promoter probe for chromatin immunoprecipitation-quantitative PCR/PCR. (H, I) Chromatin immunoprecipitation with NFAT5 antibody in BV2 cells was analyzed by PCR ( n = 3) (H) and quantitative PCR ( n = 3) (I). H3 represents the positive control group, and IgG represents the negative control. The data were presented as mean with standard deviation. ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001; ns, no statistical significance.

Article Snippet: Next, we obtained BV2 cells with stable NFAT5 knockdown by 2 μg/mL puromycin (MCE, USA) treatment.

Techniques: Binding Assay, Luciferase, Reporter Assay, Cotransfection, Control, Plasmid Preparation, Mutagenesis, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Positive Control, Negative Control, Standard Deviation

Journal: Genes & Diseases

Article Title: Microglial NFAT5 aggravates neuroinflammation via mediating NLRP6 inflammasome in experimental ischemic stroke

doi: 10.1016/j.gendis.2025.101614

Figure Lengend Snippet: NFAT5 regulates NLRP6 mRNA stability through the Nlrp6 5′UTR. (A) Relative expression of Nlrp6 mRNA in wild-type BV2 cell line after OGD/R modeling and actinomycin D (Act D) treatment for 0, 1, 2, 4, and 6 h ( n = 3). ( B ) Relative expression of Nlrp6 mRNA in sh-NC and sh-NFAT5 BV2 cell lines after OGD/R modeling and Act D treatment for 0, 1, 2, 4, and 6 h ( n = 3). ( C ) Construction of pGL-promoter Nlrp6 5′UTR plasmid containing the mouse Nlrp6 5′UTR region for dual-luciferase reporter assay. (D – F) Relative luciferase activity of pGL-promoter or pGL-promoter Nlrp6 5′UTR after co-transfection with NFAT5-overexpressing (NFAT5) or control vector (TR) into the 293T cell line. Relative luciferase activity was determined and normalized to Renilla reference luciferase activity ( n = 3). (G) Construction of pGL-promoter Nlrp6 3′UTR plasmid containing the mouse Nlrp6 3′-UTR region for dual-luciferase reporter assay. (H – J) Relative luciferase activity of pGL-promoter or pGL-promoter Nlrp6 3′UTR after co-transfection with NFAT5 overexpressing (NFAT5) or control vector (TR) into the 293T cell line ( n = 3). The relative luciferase activity was determined and normalized to Renilla reference luciferase activity. The data were presented as mean with standard deviation. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; ns, no statistical significance.

Article Snippet: Next, we obtained BV2 cells with stable NFAT5 knockdown by 2 μg/mL puromycin (MCE, USA) treatment.

Techniques: Expressing, Plasmid Preparation, Luciferase, Reporter Assay, Activity Assay, Cotransfection, Control, Standard Deviation

Journal: Genes & Diseases

Article Title: Microglial NFAT5 aggravates neuroinflammation via mediating NLRP6 inflammasome in experimental ischemic stroke

doi: 10.1016/j.gendis.2025.101614

Figure Lengend Snippet: The schematic of microglial NFAT5 aggravating neuroinflammation and neuronal injury via mediating NLRP6 inflammasome following ischemic stroke.

Article Snippet: Next, we obtained BV2 cells with stable NFAT5 knockdown by 2 μg/mL puromycin (MCE, USA) treatment.

Techniques: