Journal: PLoS ONE

Article Title: Jun Is Required in Isl1 -Expressing Progenitor Cells for Cardiovascular Development

doi: 10.1371/journal.pone.0057032

Figure Lengend Snippet: Sagittal sections of E10.5 mutant ( B, D, G ) and control ( A, C, F ) embryos analyzed by pHH3 and NFATc1 immunostaining and by TUNEL assay. pHH3 immunostaining (pink) reveals similar proliferation rates in mutant and control OFT cells ( A, B ). Dotted lines show representative areas used for cell counting with ImageJ software. TUNEL assay reveals similar numbers of TUNEL-positive (green) OFT cells in mutant and control embryos ( C, D ). ( E ) Quantitative analysis of the percentage of pHH3- and TUNEL-positive OFT cells in serial sections showing a statistically insignificant trend toward less proliferation and more apoptosis in conditionally deleted embryos at E10.5. Results are expressed as mean ± SEM of the percent positive nuclei. The statistical significance of differences between groups was analyzed by the Student’s t-test. ( F, G ) Expression of the endocardial marker NFATc1 (pink), is not significantly different between conditional mutant and control embryos. Saggital sections of anti-Tfap2a immunostained Jun flox/− ( H ) and Isl1 Cre/+ ; Jun flox/− ( I ) embryos showing no significant difference in Tfap2a-expressing neural crest cells (arrowheads) at E10.5. Sections were co-stained with DAPI to illustrate nuclei. A, atrium; AVC, atrioventricular cushion; LV, left ventricle; OFT, outflow tract; OFTC, outflow tract cushion; SMA, smooth muscle actin.

Article Snippet: Immunofluorescence (IF) and horseradish peroxidase (HRP) immunostaining were performed as described previously using rabbit monoclonal anti-Jun 60A8 (1∶100 IF; Cell Signaling Technology, Danvers, MA), rat monoclonal anti-CD31 MEC13.3 (Pecam1; 1∶100; BD Pharmingen, Franklin Lakes, NJ), mouse monoclonal anti-Isl-1 39.4D5 (1∶25 IF; Developmental Studies Hybridoma Bank, Iowa City, IA), rabbit polyclonal anti-GFP (1∶200 IF, Life Technologies), mouse monoclonal anti-SMA 1A4 (1∶200 IF, Life Technologies), rabbit polyclonal phospho-histone H3 Ser10 (1∶200 IF, Cell Signaling Technology), mouse monoclonal AP-2 alpha (Tfap2a) 3B5 (1∶4 IF, Developmental Studies Hybridoma Bank) and mouse monoclonal NFATc1 7A6 (1∶25 IF, Developmental Studies Hybridoma Bank).

Techniques: Mutagenesis, Control, Immunostaining, TUNEL Assay, Cell Counting, Software, Expressing, Marker, Staining

Journal: Journal of medicinal chemistry

Article Title: A Peptidyl Inhibitor that Blocks Calcineurin-NFAT Interaction and Prevents Acute Lung Injury

doi: 10.1021/acs.jmedchem.0c01236

Figure Lengend Snippet: Effect of CNI93 on NFATc3 nuclear translocation and cytokine secretion ex vivo. (A,B) Mouse bone marrow-derived macrophages were pretreated with 1 μM biotinylated CPP9-VAVAA (A) or CNI93 (B) and stimulated with LPS (100 ng/mL) or PBS for 1 h. Intracellular NFATc3 (green) and biotinylated peptide (red) were visualized using Nikon A1R microscope equipped with 488 nm and 543 nm excitation lasers. Single confocal optical sections (pinhole set to achieve 1 Airy unit) are shown (n = 7–10 cells/group). Scale bars, 10 μm. (C,D) Inhibition of LPS-induced TNFα and IL-6 secretion by CNI93. Total lung macrophages from healthy mice were pretreated for 4 h with indicated concentrations of DMSO, CPP9-VAVAA, CsA, or CNI93 and stimulated with LPS (100 ng/mL for 8 h) or PBS. Statistical significance determined by one-way ANOVA to vehicle (0.1% DMSO) treated cells, *, p < 0.05, **, p < 0.01, ***, p < 0.001.

Article Snippet: Antibodies for immunofluorescence and flow cytometry were purchased from the following suppliers: α-mouse CD45-PerCP/Cy5.5 and streptavidin-Alexa Fluor 594 from Biolegend (Cat. No. NC0217834 and 405240), α-rabbit IgG-Alexa Fluor 488 from Cell Signaling (Cat. No. 4412S), and Human/Mouse/Rat NFATc3 from R&D Systems (Cat. No. AF5834).

Techniques: Translocation Assay, Ex Vivo, Derivative Assay, Microscopy, Inhibition

Journal: Journal of Neuroinflammation

Article Title: SAFit2 reduces neuroinflammation and ameliorates nerve injury-induced neuropathic pain

doi: 10.1186/s12974-022-02615-7

Figure Lengend Snippet: List of used TaqMan® gene expression assays

Article Snippet: NFATc3 , Nuclear factor of activated T cells, calcineurin dependent 3 , Mm01249200_m1 , Thermo Fisher.

Techniques: Gene Expression