Journal: Toxicological sciences : an official journal of the Society of Toxicology

Article Title: Acute 4,4′-Methylene Diphenyl Diisocyanate Exposure-Mediated Downregulation of miR-206-3p and miR-381-3p Activates Inducible Nitric Oxide Synthase Transcription by Targeting Calcineurin/NFAT Signaling in Macrophages

doi: 10.1093/toxsci/kfz215

Figure Lengend Snippet: Calcineurin isozymes and Nfats mRNA levels in BALCs isolated from MDI aerosol-exposed mice. Total RNA isolated from BALCs from MDI aerosol-exposed mice and determined by RT-qPCR. Endogenous mRNA expression of (A) Ppp3ca, (B) Ppp3r1, (C) Nfatc1, and (D) Nfatc3 was determined 4 or 24h post MDI aerosol exposure (N = 3; bars, SEM). Air, house air; MDI, 4,4′-methylene diphenyl diisocyanate (**p < .01).

Article Snippet: Gene expression assays and miR specific assays used in this study were purchased from Thermo Fisher Scientific and include human PPP3CA (Hs00174223_m1) , NOS2/iNOS (Hs01075529_m1), B2M (Hs00187842_m1), mouse Ppp3ca (Mm01317678_m1), Ppp3cb (Mm00920265_m1), Ppp3cc (Mm01318938_m1), Ppp3r1 (Mm01187904_m1), Ppp3r2 (Mm01349242_g1), Nfatc1 (Mm01265944_m1), Nfatc2 (Mm01240677_m1), Nfatc3 (Mm01249200_m1), Nfatc4 (Mm00452375_m1), Nos2/iNos (Mm00440502_m1), B2m (Mm00437762_m1), mmu-miR-183–5p/hsa-miR-183–5p (Assay ID No. 002269; mmu/ Mus musculus , hsa/ Homo sapiens ), mmu-miR-206–3p/hsa-miR-206–3p (No. 000510), mmu-miR-381–3p/hsa-miR-381–3p (No. 000571), and U6 snRNA (No. 001973).

Techniques: Isolation, Aerosol, Quantitative RT-PCR, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Inhibition of Phosphoinositide 3-Kinase/Protein Kinase B Signaling Hampers the Vasopressin-dependent Stimulation of Myogenic Differentiation

doi: 10.3390/ijms20174188

Figure Lengend Snippet: HDAC4 and NFATc3 expression/cellular localization are modulated by LY294002 in AVP-treated L6 cells. L6 cells were cultured in growth medium and after 24 h were shifted in serum-free medium and treated with 0.1 μM AVP in the presence and in the absence of 20 μM LY294002 for 48 h. ( A , B ) Immunofluorescences analysis was performed to evaluate the expression levels and cellular localization of HDAC4 and NFATc3 (original magnification 20×). ( C ) Western Blot analyses of total lysates and cytosolic and nuclear fractions were achieved to evaluate changes of cellular localization of HDAC4 and NFATc3 after LY294002 addition in AVP-treated and un-treated L6 cells. ( D ) Densitometric analyses were performed using an anti-α-tubulin antibody to verify the equal loading of the samples and the cytoplasmic contamination of the nuclear fractions. Nuclear and cytosolic fractions were normalized using Stain-Free technology. Representative blots are shown. Statistical analysis was performed by Student’s t -test on data obtained from three independent experiments. * p < 0.05; ** p < 0.01.

Article Snippet: The cells were incubated overnight at 4 °C with the following primary antibodies: anti-MHC MF20 antibody (Developmental Hybridoma-Bank University of Iowa, Iowa City, IA, USA); anti-myogenin, anti- NFATc3, anti- MEF-2C, anti-HDAC4 antibodies were from Santa Cruz Biotechnology, Dallas, TX, USA; anti-FoxO3a, from Millipore, Burlington, MA, USA.

Techniques: Expressing, Cell Culture, Western Blot, Staining

Journal: International Journal of Molecular Sciences

Article Title: Inhibition of Phosphoinositide 3-Kinase/Protein Kinase B Signaling Hampers the Vasopressin-dependent Stimulation of Myogenic Differentiation

doi: 10.3390/ijms20174188

Figure Lengend Snippet: Schematic representation of signaling pathways involved in the AVP-dependent myogenic differentiation. The stimulation of AVP-V1aR signaling in myogenic cells results in the activation of the CaMK and the calcineurin pathways. The AVP-dependent combined activation of both pathways results in the formation of multifactorial complexes on the promoters of muscle-specific genes and strongly stimulates myogenic differentiation. The V1aR is a G-protein coupled receptor, whose occupancy activates PI3K. Inhibition of PI3K/Akt signaling by LY294002 results in mTOR, MEF2, and myogenin downregulation, affects the cellular localization of MEF2 preventing its nuclear translocation, upregulates the expression of FoxO3a leading to the activation of ubiquitin ligase atrogin-1, thus impinging upon the AVP-dependent myogenic differentiation. Additionally, the inhibition of the PI3K pathway by LY294002 represses the AVP-dependent HDAC4 nuclear export and, consequently, hampers the binding of multifactorial complexes (which include NFATc3 and MEF2) on the promoter region of muscle-specific genes, impinging on the full development of the differentiation program. Of note, recent evidence highlights that the Ca 2+ -dependent activation of the CaMK pathway stimulates Akt/mTOR signaling in normal and neoplastic B-lymphoid cells, suggesting a crosstalk between these signaling which may potentiate the myogenic effect of AVP (T-bars represent inhibition).

Article Snippet: The cells were incubated overnight at 4 °C with the following primary antibodies: anti-MHC MF20 antibody (Developmental Hybridoma-Bank University of Iowa, Iowa City, IA, USA); anti-myogenin, anti- NFATc3, anti- MEF-2C, anti-HDAC4 antibodies were from Santa Cruz Biotechnology, Dallas, TX, USA; anti-FoxO3a, from Millipore, Burlington, MA, USA.

Techniques: Protein-Protein interactions, Activation Assay, Inhibition, Translocation Assay, Expressing, Ubiquitin Proteomics, Binding Assay

Journal: PLoS ONE

Article Title: Fasudil improves endothelial dysfunction in rats exposed to chronic intermittent hypoxia through RhoA/ROCK/NFATc3 pathway

doi: 10.1371/journal.pone.0195604

Figure Lengend Snippet: NFATc3 protein levels were measured in aortas from Normoxia, CIH and CIH + Fa groups by Western blotting. The results were expressed as the mean ± SE. * p < 0.05, CIH group vs Normoxia group; # p < 0.05, CIH + Fa group vs CIH group (n = 6 for each group).

Article Snippet: After blocking with non-fat milk, the blots were incubated overnight at 4°C with primary antibodies for ET-1 (Abcam, ab2786, 1:600), ROCK-2 (Abcam, ab71598, 1:600), anti-eNOS (Abcam, ab50010, 1:600), anti-p-eNOS Ser 1177 (Abcam, ab195944, 1:600), anti-RhoA (Abcam, ab187027, 1:3000), anti-MYPT (Cell Signaling, 2634p, 1:1000), anti-p-MYPT (Cell Signaling, 4563p, 1:1000), anti-NFATc3 (Novus, NB100-92190, 1:500) and anti-β-actin ( Sigma , A5316, 1:1000).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Fasudil improves endothelial dysfunction in rats exposed to chronic intermittent hypoxia through RhoA/ROCK/NFATc3 pathway

doi: 10.1371/journal.pone.0195604

Figure Lengend Snippet: CIH increased RhoA, ROCK and NFATc3 protein expression in aortas, and decreased p-eNOS protein expression, which lead to endothelial dysfunction. Fasudil could improve these changes.

Article Snippet: After blocking with non-fat milk, the blots were incubated overnight at 4°C with primary antibodies for ET-1 (Abcam, ab2786, 1:600), ROCK-2 (Abcam, ab71598, 1:600), anti-eNOS (Abcam, ab50010, 1:600), anti-p-eNOS Ser 1177 (Abcam, ab195944, 1:600), anti-RhoA (Abcam, ab187027, 1:3000), anti-MYPT (Cell Signaling, 2634p, 1:1000), anti-p-MYPT (Cell Signaling, 4563p, 1:1000), anti-NFATc3 (Novus, NB100-92190, 1:500) and anti-β-actin ( Sigma , A5316, 1:1000).

Techniques: Expressing

Journal: Acta Biochimica et Biophysica Sinica

Article Title: miR-339-3p promotes AT1-AA-induced vascular inflammation by upregulating NFATc3 protein expression in vascular smooth muscle cells

doi: 10.3724/abbs.2023009

Figure Lengend Snippet: The binding sites of miR-339-3p and the 5′UTR of NFATc3 were predicted by bioinformatics analysis and verified by dual-luciferase reporter assay (A) KEGG analysis of the mRNAs whose 5′UTR could bind to miR-339-3p. (B) Prediction of the binding site between miR-339-3p and the 5′UTR of NFATc3 using the miRwalk website. The dual-luciferase reporter assay was used to verify the binding of miR-339-3p and the 5′UTR of NFATc3. (C) The mimic and inhibitor of miR-339-3p were used to upregulate and downregulate the expression of miR-339-3p in VSMCs, respectively, and the protein expression of NFATc3 was detected by western blot analysis. Data are presented as the mean±standard error. n=3 or 6. ** P<0.01, *** P<0.001, and **** P<0.0001.

Article Snippet: The inflammatory response of vascular tissue and VSMCs was detected using antibodies against NFATc3 (18222-1-AP, 1:1000 dilution; Proteintech, Wuhan, China) and IL-6 (DF6087, 1:500 dilution; Affinity Biosciences, Cincinnati, USA), IL-1β (DF6251, 1:500 dilution; Affinity Biosciences) and TNF-α (ab205587, 1:500 dilution; Abcam).

Techniques: Binding Assay, Luciferase, Reporter Assay, Expressing, Western Blot

Journal: Acta Biochimica et Biophysica Sinica

Article Title: miR-339-3p promotes AT1-AA-induced vascular inflammation by upregulating NFATc3 protein expression in vascular smooth muscle cells

doi: 10.3724/abbs.2023009

Figure Lengend Snippet: Elevated miR-339-3p is involved in the AT1-AA-induced increase in NFATc3 protein expression The expression of NFATc3 protein in (A) AT1-AA-positive rat thoracic aortas and (B) AT1-AA-treated VSMCs was detected by western blot analysis. (C) VSMCs were treated with the inhibitor of miR-339-3p and then treated with AT1-AA, and the expression of NFATc3 protein was detected by western blot analysis. Data are presented as the mean±standard error. n=5 or 6. ** P<0.01 vs the saline/vehicle/NC-inhibitor group.

Article Snippet: The inflammatory response of vascular tissue and VSMCs was detected using antibodies against NFATc3 (18222-1-AP, 1:1000 dilution; Proteintech, Wuhan, China) and IL-6 (DF6087, 1:500 dilution; Affinity Biosciences, Cincinnati, USA), IL-1β (DF6251, 1:500 dilution; Affinity Biosciences) and TNF-α (ab205587, 1:500 dilution; Abcam).

Techniques: Expressing, Western Blot, Saline

Journal: The Journal of Experimental Medicine

Article Title: The transcription factor NFATc2 controls IL-6–dependent T cell activation in experimental colitis

doi: 10.1084/jem.20072484

Figure Lengend Snippet: Enhanced expression of NFATc2 transcription factors in patients with IBDs compared with control patients. (A) Immunohistochemistry for NFATc2 expression. Colon cross sections were incubated with NFATc2-specific antibodies and analyzed by microscopy. An increased expression of NFATc2 was observed in sections from UC and CD patients compared with control patients. Representative stainings from 5 to 10 patients per group are shown. (B) Quantitative analysis of positive cells revealed a significantly increased number of NFATc2-positive cells in IBD patients compared with control patients. Data represent mean values ± the SD per high power field. *, P < 0.05. (C) Detection of NFATc2-expressing T cells in the lamina propria of patients with IBDs. Samples from CD, UC, and control patients were stained with anti-NFATc2 and anti-CD3 antibodies, followed by confocal laser microscopy. Nuclei were stained with DAPI. Representative pictures are shown. Staining with an isotype control antibody served as negative control. CD3/NFATc2 double-positive, yellowish cells were seen in the lamina propria in CD and UC. Bars: (A) 80 μm; (C) 30 μm.

Article Snippet: For staining of NFATc2 transcription factor, the mouse antibodies G1-D10 or G-20 (Santa Cruz Biotechnology) were used.

Techniques: Expressing, Control, Immunohistochemistry, Incubation, Microscopy, Staining, Negative Control

Journal: The Journal of Experimental Medicine

Article Title: The transcription factor NFATc2 controls IL-6–dependent T cell activation in experimental colitis

doi: 10.1084/jem.20072484

Figure Lengend Snippet: A regulatory role of NFATc2 in oxazolone-induced experimental colitis. (A) Enhanced expression of the NFATc2 transcription factor in oxazolone-induced colitis. Colonic cryosections from WT and NFATc2 KO mice were incubated with anti-NFATc2 antibodies followed by tyramide signal amplification. An increased expression of NFATc2 was observed in colonic tissue from WT mice with oxazolone colitis (WT oxa) as compared with WT unchallenged mice. Colonic tissue from NFATc2 KO mice (NFATc2 oxa) served as negative control and did not reveal any specific staining as expected. (B) Oxazolone colitis was induced by sensitizing mice with oxazolone, followed by intrarectal administration of the hapten reagent after 1 wk. The body weight of the mice was monitored after oxazolone rechallenge at the indicated time points. Mean values ± the SEM from two representative experiments out of six are shown. The average weight of the mice at the beginning of the experiments was 22.9 g (WT group) and 22.5 g (NFATc2 KO group), respectively. For this experiment, 6 WT and 7 NFATc2 KO mice were used. (C) Histological sections (left) of colonic inflammation in WT or NFATc2-deficient mice upon oxazolone administration. Signs of inflammation such as goblet cell depletion, ulcers, and accumulation of mononuclear cells were noted in WT mice, whereas NFATc2 KO mice showed little or no evidence of colitis. Quantitative histopathologic assessment of colitis activity (right) showed a significant (*P < 0.05) protection from inflammation and tissue injury in NFATc2 KO mice compared with WT mice. Data represent mean values ± the SEM from one representative experiment out of six. (D) High-resolution miniendoscopic analysis (left) of the colon of NFATc2 KO and WT mice in oxazolone colitis. Marked erosions and ulcers were seen in the WT group, whereas an almost normal colon architecture was noted in NFATc2-deficient mice. Quantitative endoscopic analysis (right) of inflammation (MEICS score) in WT and NFATc2 KO mice in oxazolone colitis was done at day 2 after administration of oxazolone. A significantly (P < 0.05) lower endoscopic score was observed in NFATc2-deficient mice compared with WT mice. (E) Adoptive T cell transfer from WT and NFATc2-deficient mice in SCID mice. CD4 + T cells were transferred into CB-17/SCID mice, followed by oxazolone sensitization and intrarectal oxazolone administration. The body weight of reconstituted mice was analyzed at indicated time points. Mean values ± SEM from one representative experiment out of three are shown. Whereas mice reconstituted with WT T cells showed a marked weight loss, mice given NFATc2-deficient T cells were protected from colitis and gained weight. For this experiment five SCID mice in each group were used. Bars: (A) 80 μm; (C) 100 μm.

Article Snippet: For staining of NFATc2 transcription factor, the mouse antibodies G1-D10 or G-20 (Santa Cruz Biotechnology) were used.

Techniques: Expressing, Incubation, Amplification, Negative Control, Staining, Activity Assay

Journal: The Journal of Experimental Medicine

Article Title: The transcription factor NFATc2 controls IL-6–dependent T cell activation in experimental colitis

doi: 10.1084/jem.20072484

Figure Lengend Snippet: Augmented T cell apoptosis in the colon of NFATc2-deficient mice in oxazolone-induced colitis. (A) Cryosections of colonic tissue from WT and NFATc2-deficient mice in oxazolone-induced colitis were made, and apoptotic cells were stained using the TUNEL reaction. Staining of the nuclei was done with DAPI. Representative stainings from WT and NFATc2 KO mice, as well as negative and positive control stainings are shown (left). (right) Quantitative analysis of apoptotic cells in 10 randomly selected high power fields per sample. Data represent mean values ± the SD. A significantly (P < 0.05) higher number of apoptotic cells was observed in colonic tissue from NFATc2-deficient mice in oxazolone-induced colitis compared with WT control mice. Bar, 100 μm. (B) Double staining analysis of colonic tissue from WT and NFATc2-deficient mice in oxazolone-induced colitis using TUNEL assays and anti-CD3 antibodies (left). Cryosections were analyzed by confocal laser microscopy for apoptotic T cells, and representative pictures are shown (right). A higher number of apoptotic/CD3 double-positive cells (FITC TUNEL staining; Cy3 stained CD3) was observed in NFATc2 mice compared with WT mice. (C) An increased number of apoptotic LPMCs in NFATc2-deficient mice upon administration of oxazolone. LPMCs from WT and NFATc2-deficient mice were isolated and stained with 7-AAD and anti–Annexin V antibodies for FACS analysis. In comparison to WT cells, LPMCs from NFATc2-deficient mice showed an increased number of apoptotic cells in oxazolone-induced colitis. Representative FACS analyses of different groups of mice for annexin V are shown. Quantitative analysis of apoptotic LPMCs was performed in three independent experiments (bottom). Data represent mean values ± the SD. There was a significant (*, P < 0.05) increase of apoptotic LPMCs in NFATc2-deficient mice as compared with WT control mice. (D) Increased number of apoptotic lamina propria CD4 + T cells in NFATc2-deficient mice in oxazolone-induced colitis. LPMCs from WT and NFATc2-deficient mice were prepared, and CD4 + T cells were isolated by using the MACS-System. Cells were stained with 7-AAD and anti–Annexin V antibodies for subsequent analysis (top). Lamina propria T cells from NFATc2 KO mice showed an increased percentage of apoptotic cells in colitis compared with WT mice. Quantitative analysis of apoptotic CD4 T cells was performed in two experiments (bottom). Data represent mean values ± the SD. There was a significant (*, P < 0.05) increase of apoptotic lamina propria T cells in NFATc2-deficient mice compared with WT control mice.

Article Snippet: For staining of NFATc2 transcription factor, the mouse antibodies G1-D10 or G-20 (Santa Cruz Biotechnology) were used.

Techniques: Staining, TUNEL Assay, Positive Control, Control, Double Staining, Microscopy, Isolation, Comparison

Journal: The Journal of Experimental Medicine

Article Title: The transcription factor NFATc2 controls IL-6–dependent T cell activation in experimental colitis

doi: 10.1084/jem.20072484

Figure Lengend Snippet: Cytokine production in oxazolone-induced colitis. (A) Cytokine production by splenic CD4 + T cells from WT and NFATc2-deficient mice in oxazolone-induced colitis. CD4 + T cells were stimulated with antibodies to CD3 and CD28 for 48 h, followed by analysis of culture supernatants using cytomix (see Materials and methods). Data represent mean values of four to eight mice per group. CD4 + T cells from WT mice produced significantly (P < 0.05) higher amounts of IFN-γ, but lower amounts of IL-4 than NFATc2-deficient T cells. Furthermore, a significantly lower production of IL-6 and -17 was observed in the supernatant of NFATc2-deficient CD4 + T cells in comparison to WT T cells. (B) Cytokine production by T cell–enriched lamina propria cells from WT and NFATc2-deficient mice in oxazolone-induced colitis. Lamina propria cells were stimulated with PMA/ionomycin, and the cell supernatant was analyzed using cytomix. Data represent mean values of three to six mice per group. T cells from WT mice produced higher amounts of IFN-γ, IL-6, IL-13, and IL-17 when compared with cells from NFATc2-deficient mice. At the end of the culture, cell survival rates differed by ∼10% between WT and KO cells only, suggesting that the marked differences in cytokine production under our experimental conditions are not caused by primary effects on T cell apoptosis. (C) IL-6 production by purified CD4 + lamina propria T cells from WT and NFATc2-deficient mice in oxazolone-induced colitis. LPMCs from WT and NFATc2-deficient mice were prepared, and CD4 + T cells were isolated by using the MACS System. T cells were stimulated with PMA/ionomycin, and supernatants were analyzed using an IL-6–specific ELISA. Data represent mean value ± the SD from two experiments with four mice per group. CD4 + lamina propria T cells from WT mice produced significantly (P < 0.05) higher amounts of IL-6 compared with cells from NFATc2-deficient mice.

Article Snippet: For staining of NFATc2 transcription factor, the mouse antibodies G1-D10 or G-20 (Santa Cruz Biotechnology) were used.

Techniques: Produced, Comparison, Purification, Isolation, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Experimental Medicine

Article Title: The transcription factor NFATc2 controls IL-6–dependent T cell activation in experimental colitis

doi: 10.1084/jem.20072484

Figure Lengend Snippet: Administration of hyper-IL-6 restores the susceptibility of NFATc2-deficient mice to oxazolone-induced colitis. (A) Oxazolone colitis was induced by sensitizing mice with oxazolone, followed by intrarectal administration after 7 d and monitoring of the body weight at indicated time points. Hyper IL-6 (hIL-6; 1 μg per mouse) was given i.p. before oxazolone administration. Weight curves from one representative experiment out of three are shown. Data represent mean values ± the SEM. Hyper-IL-6 administration led to colitis development and weight loss in oxazolone-treated NFATc2 KO mice, but had little effect on colitis activity in WT mice. This experiment was performed three times with groups of four to five mice. (B) Histological analysis of colonic inflammation in WT and NFATc2-deficient mice given PBS or hyper-IL-6. Signs of inflammation such as goblet cell depletion, erosions, and accumulation of mononuclear cells were noted in NFATc2-deficient mice treated with hyper-IL-6, but not control-treated NFATc2 KO mice. (C) Quantitative histopathologic assessment of colitis activity showed a significant (P < 0.05) protection from inflammation and tissue injury in NFATc2-deficient mice compared with WT mice, whereas no significant difference was noted between WT mice and NFATc2 KO mice given hyper-IL-6. Data represent mean values ± the SEM. (D) Endoscopic analysis of the colon of WT and NFATc2-deficient mice at day 2 after application of oxazolone. Oxazolone-induced erosions and ulcers were seen in the WT and NFATc2/hyper-IL-6 groups, whereas a normal colon architecture was noted in NFATc2-deficient mice. (E) Quantitative endoscopic score of inflammation (MEICS score). A significantly lower endoscopic score was observed in NFATc2-deficient mice compared with WT mice and NFATc2-deficient mice treated with hyper-IL-6. (F) IL-6 and -13 cytokine production by isolated CD4 + CD25 − splenic T cells and LPMCs in oxazolone colitis. Whereas NFATc2-deficient cells produced lower amounts of IL-6 and -13 than WT cells, hyper-IL-6 administration led to a strong induction of IL-6 and -13 production by cells lacking NFATc2. No changes in IFN-γ production were noted between NFATc2 KO mice and NFATc2 KO mice given hIL-6 (not depicted). (G) Increased numbers of CD4 + T cells in NFATc2-deficient mice after administration of hIL-6 in oxazolone colitis. Cryosections of colonic tissue were incubated with anti-CD4 antibodies and stained with conjugated Cy3 antibodies. An increased number of CD4 + T cells was observed in colonic tissue from WT mice during oxazolone-induced colitis compared with NFATc2 KO mice. After administration of hIL-6, NFATc2-deficient mice showed increased numbers of CD4 + T cells compared with NFATc2 mice without hIL-6 administration. Negative control staining is shown. Bars: (B) 100 μm; (G) 80 μm.

Article Snippet: For staining of NFATc2 transcription factor, the mouse antibodies G1-D10 or G-20 (Santa Cruz Biotechnology) were used.

Techniques: Activity Assay, Control, Isolation, Produced, Incubation, Staining, Negative Control

Journal: Advanced Science

Article Title: Inhibition of NFAT5‐Dependent Astrocyte Swelling Alleviates Neuropathic Pain

doi: 10.1002/advs.202302916

Figure Lengend Snippet: Targeting NFAT5 alleviated CCI‐induced neuropathic pain. A1‐A3) Behavioral tests of paw withdrawal mechanical threshold (PWMT), paw withdrawal thermal latency (PWTL), and spontaneous pain scores after CCI surgery. *** p < 0.001, n = 8, two‐way ANOVA with repeated measures followed by Tukey's multiple comparison test. B1‐B3) NFAT5 protein level in the injured ipsilateral spinal dorsal horn (SDH) and ipsilateral L4‐L5 DRG after sham or CCI surgery. * p < 0.05, ** p < 0.01, n = 6, one‐way ANOVA followed by Tukey's multiple comparison test. C1) Validation of knockdown efficiency after intrathecal ( i.t .) injection of AAV2/9‐sh Nfat5 in rats SDH. * p < 0.05, n = 6, two‐tailed unpaired student's t ‐test. C2‐C3) Rats were i.t . injection of AAV2/9‐sh Nfat5 for 2 weeks followed by CCI modeling and then performed PWMT and PWTL pain behavioral tests. CCI + AAV2/9‐sh Nfat5 versus CCI + AAV2/9‐NC, * p < 0.05, ** p < 0.01, *** p < 0.001, n = 8, two‐way ANOVA with repeated measures followed by Tukey's multiple comparison test. D1‐D3) Behavioral tests of PWMT, PWTL and spontaneous pain scores after daily i.t . administration of the NFAT5 inhibitor KRN2 for 7 days after CCI modeling. CCI + KRN2 (5 µg kg −1 ) versus CCI + Vehicle, * p < 0.05, n = 8, CCI + KRN2 (20 µg kg −1 ) versus CCI + Vehicle, ** p < 0.01, *** p < 0.001, n = 8, two‐way ANOVA with repeated measures followed by Tukey's multiple comparison test. E1‐E2) Behavioral tests of PWMT and PWTL after single i.t . administration of the NFAT5 inhibitor KRN2 on day 7 CCI‐treated rats. CCI + KRN2 (5 µg kg −1 ) versus CCI + Vehicle, * p < 0.05, *** p < 0.001, n = 8, CCI + KRN2 (20 µg kg −1 ) versus CCI + Vehicle, * p < 0.05, ** p < 0.01, *** p < 0.001, n = 8, two‐way ANOVA with repeated measures followed by Tukey's multiple comparison test. E3) Behavioral tests of spontaneous pain scores after single i.t . administration of the NFAT5 inhibitor KRN2 on day 7 CCI‐treated rats. CCI + KRN2 (5 µg kg −1 ) versus CCI + Vehicle, * p < 0.05, n = 8, CCI + KRN2 (20 µg kg −1 ) versus CCI + Vehicle, *** p < 0.001, n = 8, one‐way ANOVA followed by Tukey's multiple comparison test.

Article Snippet: Recombinant AURKB protein (0.1 μg, Abnova) and NFAT5 protein (1 μg, Abnova), ATP and kinase buffer (Cell Signaling Technologies) were co‐incubated for 30 min at 30°C.

Techniques: Comparison, Biomarker Discovery, Knockdown, Injection, Two Tailed Test

Journal: Advanced Science

Article Title: Inhibition of NFAT5‐Dependent Astrocyte Swelling Alleviates Neuropathic Pain

doi: 10.1002/advs.202302916

Figure Lengend Snippet: Targeting NFAT5 attenuates astrocyte swelling during neuropathic pain. A1) Representative 3D orthogonal confocal images of GFAP‐stained astrocytes in indicated group of rats SDH. Scale bar = 100 µm. A2‐A3) Morphological changes (average processes length and cell volume) of astrocytes in the SDH. CCI versus sham, ** p < 0.01, *** p < 0.001, CCI + AAV2/9‐NC versus CCI + AAV2/9‐sh Nfat5 , * p < 0.05, ** p < 0.01, n = 30 (3 rats per group, and 10 typical cells per rat were analyzed), one‐way ANOVA followed by Tukey's multiple comparison test. B1) Schematic diagram of astrocytes processes volume fraction estimation, scale bar = 10 µm. B2‐B5) Estimation of astrocytes processes volume fraction. CCI versus sham, * p < 0.05, CCI + AAV2/9‐NC versus CCI + AAV2/9‐sh Nfat5 , * p < 0.05, n = 24 (3 rats per group, and 8 volume fractions of each rat ranged from 5 to 40 µm were analyzed), two‐tailed unpaired student's t ‐test. C1) Schematic diagram of astrocytes Sholl analysis, scale bar = 10 µm. C2‐C3) Sholl analysis of astrocytes. CCI versus sham, * p < 0.05, ** p < 0.01, n = 3, CCI + AAV2/9‐NC versus CCI + AAV2/9‐sh Nfat5 , * p < 0.05, n = 3, two‐way ANOVA followed by Tukey's multiple comparison test. D) Transfection efficiency of intra‐spinal injection of AAV2/5‐sh Nfat5 in the indicated cell types. Quantification of transfection efficiency was shown on the right (pooled from 3 rats/group). Scale bar = 100 µm. E1‐E2) Representative transmission electron microscopy images and average area of the somatic cytoplasm statistics of astrocytes in indicated group of rats SDH after intra‐spinal injection of AAV2/5‐sh Nfat5 . The area within the blue curve indicates astrocytes, the white arrow in the upper panel image indicates the area that is magnified in the lower panel of the image, the yellow arrow indicates cytoplasm or mitochondria without swelling, and the red arrow indicates cytoplasm or mitochondria with obvious swelling. Scale bar = 2 µm. CCI versus sham, ** p < 0.01, CCI + AAV2/5‐NC versus CCI + AAV2/5‐sh Nfat5 , * p < 0.05, n = 3, one‐way ANOVA followed by Tukey's multiple comparison test.

Article Snippet: Recombinant AURKB protein (0.1 μg, Abnova) and NFAT5 protein (1 μg, Abnova), ATP and kinase buffer (Cell Signaling Technologies) were co‐incubated for 30 min at 30°C.

Techniques: Staining, Comparison, Two Tailed Test, Transfection, Injection, Transmission Assay, Electron Microscopy

Journal: Advanced Science

Article Title: Inhibition of NFAT5‐Dependent Astrocyte Swelling Alleviates Neuropathic Pain

doi: 10.1002/advs.202302916

Figure Lengend Snippet: NFAT5 regulates AQP4 expression. A) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of ChIP‐seq of NFAT5 targets. B) Heat map showed 144 significantly up‐regulated genes in the SDH of CCI group rats compared with the sham group by RNA‐seq analysis. C1) The intersection of NFAT5 ChIP‐seq data with RNA‐seq data from sham and CCI groups. C2) ChIP‐qPCR analysis of endogenous NFAT5 binding at Aqp4 promoters in the SDH after CCI. *** p < 0.001, n = 3, two‐tailed unpaired Student's t ‐test. C3) Expression of Aqp4 mRNA in the injured ipsilateral SDH after sham or CCI surgery. Compared to the sham‐operated rats, *** p < 0.001, n = 6, one‐way ANOVA followed by Tukey's multiple comparison test. D) Immunofluorescence double‐labeling of AQP4 (green) and astrocyte marker GFAP (red) in the SDH on day 7 after CCI modeling. Scale bar = 100 µm. E1) Aqp4 mRNA expression after i.t . administration of AAV2/9‐sh Nfat5 in the SDH of CCI rats. CCI versus sham, *** p < 0.001, CCI + AAV2/9‐sh Nfat5 versus CCI + AAV2/9‐NC, *** p < 0.001, n = 6, one‐way ANOVA followed by Tukey's multiple comparison test. E2) AQP4 protein expression after i.t . administration of AAV2/9‐sh Nfat5 in the SDH of CCI rats. CCI versus sham, ** p < 0.01, CCI + AAV2/9‐sh Nfat5 versus CCI + AAV2/9‐NC, ** p < 0.01, n = 6, one‐way ANOVA followed by Tukey's multiple comparison test.

Article Snippet: Recombinant AURKB protein (0.1 μg, Abnova) and NFAT5 protein (1 μg, Abnova), ATP and kinase buffer (Cell Signaling Technologies) were co‐incubated for 30 min at 30°C.

Techniques: Expressing, ChIP-sequencing, RNA Sequencing, ChIP-qPCR, Binding Assay, Two Tailed Test, Comparison, Immunofluorescence, Labeling, Marker

Journal: Advanced Science

Article Title: Inhibition of NFAT5‐Dependent Astrocyte Swelling Alleviates Neuropathic Pain

doi: 10.1002/advs.202302916

Figure Lengend Snippet: AURKB regulates NFAT5 phosphorylation and its protein level. A) Expression of Nfat5 mRNA in the injured ipsilateral SDH after sham or CCI surgery. B1‐B2) Co‐immunoprecipitation of AURKB and NFAT5 in the SDH of rats after CCI modeling. C) Experimental validation of AURKB regulation of NFAT5 phosphorylation in vitro. D1‐D2) Behavioral tests of PWMT and PWTL after daily i.t . administration of AURKB inhibitor AZD1152 over 7 days post‐CCI modeling. * p < 0.05, ** p < 0.01, *** p < 0.001, n = 6, two‐way ANOVA with repeated measures followed by Tukey's multiple comparison test. E1) Nfat5 mRNA expression in the SDH after daily i.t . administration of AURKB inhibitor AZD1152 in CCI rats. E2) NFAT5 total protein expression in the SDH after daily i.t . administration of AURKB inhibitor AZD1152 in CCI rats. CCI versus sham, * p < 0.05, CCI + AZD1152 versus CCI + Vehicle, * p < 0.05, n = 4, one‐way ANOVA followed by Tukey's multiple comparison test. F1) Cytoplasm and nucleus NFAT5 protein levels after daily i.t . administration of AURKB inhibitor AZD1152 in the SDH of CCI rats. CCI versus sham, * p < 0.05, CCI + AZD1152 versus CCI + Vehicle, * p < 0.05, n = 4, one‐way ANOVA followed by Tukey's multiple comparison test. F2) Nuclear to cytoplasmic ratio of NFAT5 protein of F1, * p < 0.05, CCI + AZD1152 versus CCI + Vehicle, * p < 0.05, n = 4, one‐way ANOVA followed by Tukey's multiple comparison test.

Article Snippet: Recombinant AURKB protein (0.1 μg, Abnova) and NFAT5 protein (1 μg, Abnova), ATP and kinase buffer (Cell Signaling Technologies) were co‐incubated for 30 min at 30°C.

Techniques: Phospho-proteomics, Expressing, Immunoprecipitation, Biomarker Discovery, In Vitro, Comparison

Journal: Advanced Science

Article Title: Inhibition of NFAT5‐Dependent Astrocyte Swelling Alleviates Neuropathic Pain

doi: 10.1002/advs.202302916

Figure Lengend Snippet: AURKB regulates NFAT5‐AQP4 signaling pathway in astrocytes. A1) Aqp4 mRNA expression in the SDH of CCI rats after daily i.t . administration of AURKB inhibitor AZD1152. CCI versus sham, *** p < 0.001, CCI+ AZD1152 versus CCI+ Vehicle, *** p < 0.001, n = 6, one‐way ANOVA followed by Tukey's multiple comparison test. A2) AQP4 protein expression in the SDH of CCI rats after daily i.t . administration of AURKB inhibitor AZD1152. CCI versus sham, * p < 0.05, CCI + AZD1152 versus CCI + Vehicle, ** p < 0.01, n = 4, one‐way ANOVA followed by Tukey's multiple comparison test. B1) Experimental procedure involved the overexpression of AURKB in CTX‐TNA2 cell line, followed by subsequent administration of the AURKB inhibitor AZD1152 or NFAT5 inhibitor KRN2. B2) The AURKB protein level in the LV‐ Aurkb transfected CTX‐TNA2 cell line. LV‐ Aurkb versus LV‐NC, ** p < 0.01, n = 6, one‐way ANOVA followed by Tukey's multiple comparison test. C1‐C2) The Nfat5 mRNA and protein levels in the LV‐ Aurkb transfected CTX‐TNA2 cells with AURKB inhibitor AZD1152 treatment for 24 h. Control versus LV‐ Aurkb , * p < 0.05, LV‐ Aurkb + Vehicle versus LV‐ Aurkb + AZD1152, ** p < 0.01, n = 4, one‐way ANOVA followed by Tukey's multiple comparison test. C3) The AQP4 protein levels in the LV‐ Aurkb transfected CTX‐TNA2 cells administration with AURKB inhibitor AZD1152 treatment for 24 h. Control versus LV‐ Aurkb , * p <0.05, LV‐ Aurkb + Vehicle versus LV‐ Aurkb + AZD1152, *** p < 0.001, n = 6, one‐way ANOVA followed by Tukey's multiple comparison test. D) The AQP4 protein levels in the LV‐ Aurkb transfected CTX‐TNA2 cells administration with NFAT5 inhibitor KRN2 treatment for 24 h. Control versus LV‐ Aurkb , * p <0.05, LV‐ Aurkb + Vehicle versus LV‐ Aurkb + KRN2, * p < 0.05, n = 6, one‐way ANOVA followed by Tukey's multiple comparison test. E) NFAT5 protein level in cytoplasm and nucleus of AURKB overexpressed CTX‐TNA2 cells, followed with administered AURKB inhibitor AZD1152. Control versus LV‐ Aurkb , ** p <0.01, LV‐ Aurkb + Vehicle versus LV‐ Aurkb + AZD1152, *** p < 0.001, n = 4, one‐way ANOVA followed by Tukey's multiple comparison test.

Article Snippet: Recombinant AURKB protein (0.1 μg, Abnova) and NFAT5 protein (1 μg, Abnova), ATP and kinase buffer (Cell Signaling Technologies) were co‐incubated for 30 min at 30°C.

Techniques: Expressing, Comparison, Over Expression, Transfection, Control

Journal: Advanced Science

Article Title: Inhibition of NFAT5‐Dependent Astrocyte Swelling Alleviates Neuropathic Pain

doi: 10.1002/advs.202302916

Figure Lengend Snippet: Schematic diagram of the NFAT5 signaling pathway regulation in SDH astrocytes during neuropathic pain. Peripheral nerve injury induces morphological changes in SDH astrocytes, characterized by significant cell swelling, a prominent pathological feature in the context of neuropathic pain. The NFAT5 signaling pathway plays a crucial role in astrocyte swelling after peripheral nerve injury. AURKB mediates NFAT5 phosphorylation, promotes NFAT5 translocation into the nucleus and protects it from degradation. The nuclear NFAT5 further stimulates the transcription and expression of AQP4, which subsequently leads to astrocyte swelling and contributes to the development of neuropathic pain.

Article Snippet: Recombinant AURKB protein (0.1 μg, Abnova) and NFAT5 protein (1 μg, Abnova), ATP and kinase buffer (Cell Signaling Technologies) were co‐incubated for 30 min at 30°C.

Techniques: Phospho-proteomics, Translocation Assay, Expressing