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: Developmental Biology

Article Title: Calcium/NFAT signalling promotes early nephrogenesis

doi: 10.1016/j.ydbio.2011.01.033

Figure Lengend Snippet: NFAT genes are expressed during kidney development. A) Quantitative real-time RT-PCR analysis, relative to GAPDH, of E11.5–15.5 whole kidneys reveals that all five NFAT genes are expressed. The main graph shows data on the calcium-regulated NFAT genes ( NFATc1-c4 ); the inlay graph also includes data on the non-calcium-regulated NFAT5 gene. B) Wt1-GFP (green) fluorescence in an embryonic kidney marking the MM and its derivatives. C) Expression analysis of NFATc genes in MM (Wt1-GFP positive) and whole kidney. Expression of D) NFATc3 and E) NFATc4 was confirmed by in situ hybridisation in E13.5 whole kidneys. G — gonad; K — kidney.

Article Snippet: This is in agreement with data in the Human Protein Atlas showing NFATc3 in adult renal tubules and glomeruli ( http://www.proteinatlas.org ; ( )).

Techniques: Quantitative RT-PCR, Fluorescence, Expressing, In Situ, Hybridization

Journal: Developmental Biology

Article Title: Calcium/NFAT signalling promotes early nephrogenesis

doi: 10.1016/j.ydbio.2011.01.033

Figure Lengend Snippet: NFATc3 and NFATc4 expression. 70 μm vibratome sections of E13.5 kidneys previously stained whole mount for A–B) NFATc3 and C) NFATc4 expression by in situ hybridisation. MM — metanephric mesenchyme; RV — renal vesicle; SB — s-shaped body; UB — ureteric bud.

Article Snippet: This is in agreement with data in the Human Protein Atlas showing NFATc3 in adult renal tubules and glomeruli ( http://www.proteinatlas.org ; ( )).

Techniques: Expressing, Staining, In Situ, Hybridization

Journal: Developmental Biology

Article Title: Calcium/NFAT signalling promotes early nephrogenesis

doi: 10.1016/j.ydbio.2011.01.033

Figure Lengend Snippet: NFATc3 is present in the developing nephrons and UB. Immunoperoxidase antibody detection (brown) on sections of A–C) E12.5 and D–F) E14.5 kidneys, and G–I) fluorescent antibody detection (green) on E11.5 + 72 h cultured kidney rudiments. In (A–F) cell nuclei are stained blue with haematoxylin; in (G–I) nuclei are DAPI-stained blue. Higher magnification images are presented from separate kidneys to increase the range of data shown. CB — comma-shaped body; RV — renal vesicle; SB — s-shaped body; Tu — tubule; UB — ureteric bud. Black arrows: condensed MM.

Article Snippet: This is in agreement with data in the Human Protein Atlas showing NFATc3 in adult renal tubules and glomeruli ( http://www.proteinatlas.org ; ( )).

Techniques: Cell Culture, Staining

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