Journal: The Journal of Experimental Medicine

Article Title: Counter-regulation of T cell effector function by differentially activated p38

doi: 10.1084/jem.20131917

Figure Lengend Snippet: p38 Alternative activation is required for TCR-induced expression of NFATc1 and IRF4 in CD4 + T cells. (A) Purified CD4 + T cells were stimulated with anti-CD3/CD28 in the presence or absence of SB203580 and immunoblotted for the indicated transcription factors 48 h later. (B and C) Purified CD4 + T cells were stimulated with anti-CD3/CD28 in the presence of 11R-VIVIT or 11R-VEET. Irf4 mRNA (B) and protein (C) were determined 24 h later. (D and E) Purified CD4 + T cells from WT and DKI mice were stimulated with anti-CD3/CD28 or PMA plus ionomycin, as indicated for the indicated times and analyzed for expression of Nfatc1 and Irf4 mRNA (D) and protein (E). Results shown in A–E are representative of three independent experiments each. (F) Naive CD4 + T cells were stimulated with either anti-CD3/CD28 or PMA and ionomycin under neutral or Th17-skewing conditions for 3 d. IL-17A and IFN-γ expression were determined by intracellular staining and flow cytometry after restimulation with PMA and ionomycin. Results shown in the left panel are representative of three independent experiments and bar graphs in the right panel show the mean ± SEM of all three. *, P < 0.05 (unpaired two-tailed Student’s t test).

Article Snippet: Recombinant full-length NFATc1 was purchased from SignalChem.

Techniques: Activation Assay, Expressing, Purification, Staining, Flow Cytometry, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Counter-regulation of T cell effector function by differentially activated p38

doi: 10.1084/jem.20131917

Figure Lengend Snippet: MAPK-cascade activated p38 inhibits functions distal to alternatively activated p38. (A) Purified CD4 + T cells were cultured overnight without stimulation, and then treated with either UV (50 J/m 2 or 100 J/m 2 or 200 J/m 2 ), 0.6 M sorbitol for 30 min, or PMA (10 ng/ml) and ionomycin (1 µg/ml) for 1 h. Cell lysates were immunoblotted with anti-phospho-p38. The results are representative of three independent experiments. (B) Purified CD4 + T cells were treated with either 0.6 M sorbitol for 30 min, 50 J/m 2 UV, or PMA and ionomycin were stimulated with anti-CD3/CD28 for 24 h. The expression of Nfatc1 and Irf4 was determined by quantitative real-time PCR. Results are the mean ± SEM of three independent experiments. *, P < 0.05 (unpaired two-tailed Student’s t test). (C) Cells were treated as in B, and IRF4 expression was determined by Western blot. The results are representative of two independent experiments. (D) Freshly purified T cells were stimulated or not with anti-CD3/CD28 for 48 h. Cytosolic and nuclear fractions were separated in a low percentage SDS-PAGE (8%) gel and immunoblotted for NFATc1. The results are representative of three independent experiments. (E) CD4 + T cells were stimulated for 48 h with anti-CD3/CD28, and then treated with sorbitol in the presence or absence of SB203580 and/or SP600125 for 30 min, rested for 1 h, then immunoblotted for NFATc1 in cytosolic and nuclear fractions. Results are representative of three independent experiments. (F) Purified CD4 + T cells were treated or not with sorbitol for 30 min and stimulated with anti-CD3/CD28 for 48 h, and then immunoblotted for NFATc1 in cytosolic and nuclear fractions. The results are representative of three independent experiments. (G) Jurkat T cells were stimulated for 48 h with an agonistic anti-TCR antibody (C305), and then treated with sorbitol in the presence or absence of SB203580 and/or SP600125 for 30 min. After 1 h without further stimulation, cytosolic fractions were immunoblotted for phospho-NFATc1 (p-NFATc1). The results are representative of three independent experiments. (H) Recombinant p38 was activated with Zap70, MKK6, or buffer alone in in vitro kinase buffer. After 1 h, recombinant ATF2 and 10 µCi [ 32 P]ATP were added for 30 min before separation on SDS-PAGE and PhosphorImager analysis. The phosphorylation state of p38 Y323 was determined by immunoblotting. The results are representative of three independent experiments. (I) Recombinant p38 was activated with Zap70, MKK6, or buffer alone and an in vitro kinase assay using recombinant NFATc1 as substrate was performed as in (H). The results are representative of two independent experiments. (J) Recombinant p38 was activated with MKK6 or buffer alone in in vitro kinase buffer. Active JNK1 was kept in in vitro kinase buffer. After 1 h, recombinant NFATc1 was added and incubated for an additional hour before separation on SDS-PAGE and immunoblotting with an antibody specific for NFATc1 phosphorylated on residue S172. The results are representative of two independent experiments. (K) Recombinant p38 was activated with Zap70, MKK6, or buffer alone in an in vitro kinase assay with recombinant NFATc1 as the p38 substrate, as in I. Samples were separated on a low percentage SDS-PAGE (8%) and immunoblotted for NFATc1 phosphorylated on residue S172. The results are representative of three independent experiments.

Article Snippet: Recombinant full-length NFATc1 was purchased from SignalChem.

Techniques: Purification, Cell Culture, Expressing, Real-time Polymerase Chain Reaction, Two Tailed Test, Western Blot, SDS Page, Recombinant, In Vitro, Phospho-proteomics, Kinase Assay, Incubation, Residue

Journal: Molecular Genetics & Genomic Medicine

Article Title: Genetic and functional analyses detect one pathological NFATC1 mutation in a Chinese tricuspid atresia family.

doi: 10.1002/mgg3.1771

Figure Lengend Snippet: (a) Pedigree of the family involved in the study. Black arrow represents the proband who carried tricuspid atresia. (b) Sanger sequencing confirmed that the proband carried a NFATC1 mutation which c.964G changed to A. (c and d) Fetal echocardiography of the proband indicated right ventricular dysplasia and tricuspid atresia

Article Snippet: pCMV3.1‐NFATC1 expression vector containing human NFATC1 coding sequence was purchased from Sino Biological (catalog: HG13963‐CF).

Techniques: Sequencing, Mutagenesis

Journal: Molecular Genetics & Genomic Medicine

Article Title: Genetic and functional analyses detect one pathological NFATC1 mutation in a Chinese tricuspid atresia family.

doi: 10.1002/mgg3.1771

Figure Lengend Snippet: Detailed information of candidate gene mutations

Article Snippet: pCMV3.1‐NFATC1 expression vector containing human NFATC1 coding sequence was purchased from Sino Biological (catalog: HG13963‐CF).

Techniques: Software

Journal: Molecular Genetics & Genomic Medicine

Article Title: Genetic and functional analyses detect one pathological NFATC1 mutation in a Chinese tricuspid atresia family.

doi: 10.1002/mgg3.1771

Figure Lengend Snippet: Functional experiments of NFATC1. (a) Image of western blotting. (b) Analysis of gray value of western blotting, ** p < 0.01 (c) Relative luciferase activity of DEGS1 promoter raised by NFATC1‐WT, NFATC1‐p.D322N, and control group. ** p < 0.01

Article Snippet: pCMV3.1‐NFATC1 expression vector containing human NFATC1 coding sequence was purchased from Sino Biological (catalog: HG13963‐CF).

Techniques: Functional Assay, Western Blot, Luciferase, Activity Assay

Journal: Science advances

Article Title: Matrix-bound nanovesicles alleviate particulate-induced periprosthetic osteolysis.

doi: 10.1126/sciadv.adn1852

Figure Lengend Snippet: Fig. 3. MBVs mitigate RANKL-induced osteoclast formation and functions by suppressing NF-κB pathway in vitro. RAW 264.7 cells were challenged with RANKL and various concentrations of MBV (1× 1010 to 2× 1010 particles/ml, MBV versus cells ratio 1× 104 to 2 × 104 particles/ml per cell), 1 day for osteoclast differentiation and activity– related gene RT-qPCR (A and B; RANKL, 50 ng/ml), 3 days for osteoclast differentiation and activity–related protein Western blot (C; RANKL, 50 ng/ml), and 5 days for TNF-α concentration in conditional media (D; RANKL, 30 ng/ml; MBV, 5× 109 particles/ml). (A) Osteoclast differentiation regulators (NFATc1 and DC-STAMP) relative expression (2−△△T) were measured by RT-qPCR (values = means ± SD; n = 9; significant differences, *P < 0.05). (B) Osteoclast activity–related genes (cathepsinK, c-Src, MMP-9, and β3-integrin) relative expressions (2−△△T) were measured by RT-qPCR (values = means ± SD; n = 9; significant differences, *P < 0.05). (C) Osteoclast differentiation–related protein (NFATc1) and osteoclast activity proteins (c-Src and cathepsin K) relative expression was measured by Western blot (values = means ± SD; n = 3; significant differ- ences, *P < 0.05). (D) TNF-α in conditional media from Raw 264.7 cells cocultured with RANKL with or without MBV for 5 days was determined by ELISA (values = means ± SD; n = 3; significant differences, *P < 0.05). (E) RAW 264.7 cells were challenged with RANKL (50 ng/ml) and MBV (2 × 1010 particles/ml) from 0 to 60 min. NF-κB pathway proteins were determined by Western blot. Representative Western blotting images of the effects of MBV on p-p65, p65, and IĸB-α. Quantification of the ratios of band intensity of p-p65/p65 and IĸBα relative to β-actin expression (values = means ± SD; n = 3; significant differences, *P < 0.05).

Article Snippet: Then, membranes were blocked with 5% nonfat milk in tris- buffered saline containing 0.1% Tween- 20 (TBST) for 1 hour at room temperature, and subsequently probed with 1:1000 dilution primary antibodies NFATc1 (8032S,Cell Signaling Technology, USA), cathepsin K (57056S,Cell Signaling Technology, USA), c- Src (25978- 1- AP, Proteintech, USA), phospho- p65 (3033S, Cell Signaling Technology, USA), p65 (8242S, Cell Signaling Technology, USA), IκBα (4812S, Cell Signaling Technology, USA), and βactin (4970T, Cell Signaling Technology, USA) in TBST and 1% bovine serum albumin (BSA) overnight at 4°C.

Techniques: In Vitro, Activity Assay, Quantitative RT-PCR, Western Blot, Concentration Assay, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Science advances

Article Title: Matrix-bound nanovesicles alleviate particulate-induced periprosthetic osteolysis.

doi: 10.1126/sciadv.adn1852

Figure Lengend Snippet: Fig. 7. Schematic illustration of the mechanism by which MBVs reduce particulate-induced osteolysis. MBVs were derived from a porcine urinary bladder extracel- lular matrix (UBM) bioscaffold. In vitro, MBVs reduce monocyte/macrophage differentiation to osteoclast by suppressing the NF-κB pathway and its downstream NFATc1, DC-STAMP, c-Src, and cathepsin K expression. In vivo, MBVs alleviate UHMWPE particle-induced osteolysis in a murine calvarial osteolysis model.

Article Snippet: Then, membranes were blocked with 5% nonfat milk in tris- buffered saline containing 0.1% Tween- 20 (TBST) for 1 hour at room temperature, and subsequently probed with 1:1000 dilution primary antibodies NFATc1 (8032S,Cell Signaling Technology, USA), cathepsin K (57056S,Cell Signaling Technology, USA), c- Src (25978- 1- AP, Proteintech, USA), phospho- p65 (3033S, Cell Signaling Technology, USA), p65 (8242S, Cell Signaling Technology, USA), IκBα (4812S, Cell Signaling Technology, USA), and βactin (4970T, Cell Signaling Technology, USA) in TBST and 1% bovine serum albumin (BSA) overnight at 4°C.

Techniques: Derivative Assay, In Vitro, Expressing, In Vivo

Journal: Inflammation Research

Article Title: Rhein-attenuates LPS-induced acute lung injury via targeting NFATc1/Trem2 axis

doi: 10.1007/s00011-023-01746-8

Figure Lengend Snippet: The central coordinates of protein and docking box parameters

Article Snippet: The NFATc1 inhibitor NFATc1-IN-1 was purchased from MedChemExpress (MCE, HY-147369, USA).

Techniques:

Journal: Inflammation Research

Article Title: Rhein-attenuates LPS-induced acute lung injury via targeting NFATc1/Trem2 axis

doi: 10.1007/s00011-023-01746-8

Figure Lengend Snippet: Rhein plays its anti-inflammatory role by interacting with NFATc1, the upstream transcription factor of Trem2. A Screening strategy of target transcription factor (TF). B TPM of screened TFs. C 2D interaction diagram of NFATc1-rhein. D Localized views of NFATc1-rhein composites obtained by molecular docking. E Chemical structure of rhein and biotin-labeled rhein (Bio-Rhein). F Biotin pull-down assay using RAW264.7 lysates. G qRT-PCR analysis of Trem2 expression in RAW264.7 cells transfected with si-NC or si-NFATc1 ( n = 3 per group). F Mouse NFATc1 binding motif (MA0624.2 from JASPAR database). G Diagram of binding sites of NFATc1 on Trem2 promoter region. Genomic PCR was performed using primer sets for site (− 521 bp to − 360 bp) in the Trem2 promoter. J ChIP analysis of NFATc1 enrichment of Trem2 promoter in RAW264.7 cells transfected with mock or NFATc1-V5. K Sequences of wild-type Trem2 promoter and mutant promoter region. Trem2 promoter reporter activities were evaluated in RAW264.7 cells transfected with NFATc1 expression vector. L Representative images of DHE assay to measure the intracellular ROS level (scale bar = 50 μm; n = 6 per group). M Representative images of immunofluorescence staining by labeling CD86 (red), Trem2 (green), and nuclei (DAPI, grey) (scale bar = 25 μm; n = 6 per group). N Transcription activity of Trem2 measured by Trem2 luciferase ( n = 3 per group). O–Q Quantifications of MFI of L and M . * P < 0.05. means ± SD (Color figure online)

Article Snippet: The NFATc1 inhibitor NFATc1-IN-1 was purchased from MedChemExpress (MCE, HY-147369, USA).

Techniques: Labeling, Pull Down Assay, Quantitative RT-PCR, Expressing, Transfection, Binding Assay, Mutagenesis, Plasmid Preparation, Immunofluorescence, Staining, Activity Assay, Luciferase

Journal: Inflammation Research

Article Title: Rhein-attenuates LPS-induced acute lung injury via targeting NFATc1/Trem2 axis

doi: 10.1007/s00011-023-01746-8

Figure Lengend Snippet: Small molecule-protein binding affinity evaluation based on AutoDock Vina (kcal/mol)

Article Snippet: The NFATc1 inhibitor NFATc1-IN-1 was purchased from MedChemExpress (MCE, HY-147369, USA).

Techniques: Binding Assay

Journal: Inflammation Research

Article Title: Rhein-attenuates LPS-induced acute lung injury via targeting NFATc1/Trem2 axis

doi: 10.1007/s00011-023-01746-8

Figure Lengend Snippet: Blockage of NFATc1 alleviated the anti-inflammatory role of rhein in ALI/ARDS in vivo by downregulating Trem2 expression, followed by decreased macrophage M2 polarization transition. A Representative images of H&E stained lung tissues with zoomed views below (upper scale bar = 100 μm; lower scale bar = 50 μm; yellow dotted box indicating the zoomed area; n = 6 per group). B Representative images of immunofluorescence staining labeling via Trem2 (red) and nuclei (DAPI, blue) (scale bar = 100 μm; n = 6 per group) C Wet-to-dry (W/D) lung weight ratio ( n = 6 per group). D Evans blue index ( n = 6 per group). E, F Concentrations of TNF-α and IL-1β in the bronchoalveolar lavage fluid (BALF) ( n = 6 per group). G Representative images of immunofluorescence staining labeling M1 macrophages via CD68 (green), CD86 (red), and nuclei (DAPI, blue) (upper scale bar = 100 μm; lower scale bar = 50 μm; n = 6 per group). H Representative images of immunofluorescence staining labeling M2 macrophages via CD68 (green), CD206 (red), and nuclei (DAPI, blue) (upper scale bar = 100 μm; lower scale bar = 50 μm; n = 6 per group). I, J Concentrations of IL-4 and IL-10 in the bronchoalveolar lavage fluid (BALF) ( n = 6 per group). K, L Statistics of the proportion of M1 and M2 macrophages, indicated by CD68 + CD86 + cells and CD68 + CD206 + cells versus total CD68 + cells ( n = 6 per group). * P < 0.05. means ± SD (Color figure online)

Article Snippet: The NFATc1 inhibitor NFATc1-IN-1 was purchased from MedChemExpress (MCE, HY-147369, USA).

Techniques: In Vivo, Expressing, Staining, Immunofluorescence, Labeling

Journal: Inflammation Research

Article Title: Rhein-attenuates LPS-induced acute lung injury via targeting NFATc1/Trem2 axis

doi: 10.1007/s00011-023-01746-8

Figure Lengend Snippet: Schematic diagram of the study. The upper part of the diagram indicates the overall effect of rhein administration in ALI/ARDS, including decreasing M1 macrophages, upregulated M2 polarization transition, as well as alleviated lung injury and the epithelial cells apoptosis. The lower part of the diagram demonstrates the role rhein played with macrophages, including interacting with NFATc1 and activating the NFATc1/Trem2 axis, to promote the M2 polarization transition under ALI/ARDS microenvironment

Article Snippet: The NFATc1 inhibitor NFATc1-IN-1 was purchased from MedChemExpress (MCE, HY-147369, USA).

Techniques:

Journal: Life sciences

Article Title: Selective serotonin reuptake inhibitors (SSRI) affect murine bone lineage cells

doi: 10.1016/j.lfs.2020.117827

Figure Lengend Snippet: Bone Cell Lineage Targets

Article Snippet: Low Sertraline = 34.2 ng/ml High Sertraline = 342 ng/ml n=3 assays/cell type *p≤0.05 **p≤0.01 ***p≤0.001 as compared to control table ft1 table-wrap mode="anchored" t5 Table 2: caption a7 Target TaqMan Gene Expression Assay Osteoblast Runx2 Mm00501584_m1 Alp Mm00475834_m1 Msx2 Mm00442992_m1 Dlx5 Mm00438430_m1 Osx Mm04209856_m1 Bglap Mm01741771_g1 Col1a1 Mm00801666_g1 Col1a2 Mm00483888_m1 Osteoclast Mcp-1 Mm00442991_m1 Rantes Mm01302427_m1 Rank Mm00437132_m1 Nfatc1 Mm01265944_m1 Ctsk Mm00484039_m1 Mmp9 Mm00442991_m1 Calcr Mm00432282_m1 Trap Mm00475698_m1 Open in a separate window Bone Cell Lineage Targets Osteoblast and Osteoclast Functional Assays To determine the specific effect of sertraline on bone lineage cells, pre-osteoblasts, osteoblasts, pre-osteoclasts and osteoclasts were seeded in triplicate into 96 well plates at a density of 4,000 cells per well.

Techniques: Gene Expression