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: Cell Death & Disease

Article Title: Hypoxia-induced NFATc3 deSUMOylation enhances pancreatic carcinoma progression

doi: 10.1038/s41419-022-04779-9

Figure Lengend Snippet: a Expression and location of NFATc3 in 60 samples of human PDAC tissues and matched adjacent normal tissues by IHC staining with an anti-NFATc3 antibody. Representative images are shown. Scale bars, 10 μm. b Comparative analysis of nuclear NFATc3 expression between PDAC tissues and matched adjacent normal tissues (*** P < 0.001). c Correlations between nuclear NFATc3 expression levels and PDAC clinicopathological parameters. d Kaplan–Meier plots and p -values of the log-rank test for comparing survivals of PDAC patients with high (staining score, 5–12) and low (staining score, 0–4) expression of nuclear NFATc3.

Article Snippet: Antibody against NFATc3 (A6666) was obtained from Abclonal.

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Cell Death & Disease

Article Title: Hypoxia-induced NFATc3 deSUMOylation enhances pancreatic carcinoma progression

doi: 10.1038/s41419-022-04779-9

Figure Lengend Snippet: a PANC-1 and AsPC-1 cells were stimulated with or without hypoxia for 12 h. Immunofluorescent analyses were performed with an anti-NFATc3 antibody, and the percentage of nuclear NFATc3 was quantitated (right) using ImageJ. Scale bars, 10 μm. b PANC-1 and AsPC-1 cells were stimulated with or without hypoxia for the indicated time. Cytosolic and nuclear fractions were prepared, and immunoblotting analyses with the indicated antibodies were performed. c PANC-1 and AsPC-1 cells were transfected with luciferase reporter gene plasmid (NFATc3-Luc) and were stimulated with or without hypoxia for 12 h. The data are presented as the mean ± SD of triplicate samples (*** P < 0.001). d PANC-1 and AsPC-1 cells were stimulated with or without hypoxia for the 12 h. Immunoprecipitation with an anti-NFATc3 antibody was performed. e PANC-1 and AsPC-1 cells were stimulated with or without hypoxia for the indicated time. Immunoblot analyses were performed with the indicated antibodies.

Article Snippet: Antibody against NFATc3 (A6666) was obtained from Abclonal.

Techniques: Western Blot, Transfection, Luciferase, Plasmid Preparation, Immunoprecipitation

Journal: Cell Death & Disease

Article Title: Hypoxia-induced NFATc3 deSUMOylation enhances pancreatic carcinoma progression

doi: 10.1038/s41419-022-04779-9

Figure Lengend Snippet: a , b , c , e , g Immunoprecipitation and immunoblot analyses were performed with the indicated antibodies. a AsPC-1 cells were stimulated with or without hypoxia for 12 h. b , c HEK293T cells ( b ) and AsPC-1 cells ( c ) were transfected with Flag-NFATc3, V5-SUMO1,V5-SUMO2, or V5-SUMO3 plasmids as indicated. d Sketch of NFATc3 Structural Features (upper panel). NHR, NFAT homology region; RHR, Rel homology region; TAD, transactivation domain. The potential SUMOylation sites in NFATc3 were shown (lower panel). e Flag-WT NFATc3 or mutants (384, 434, 703, and 1013) were expressed in HEK293T cells. f Lys384 of NFATc3 is evolutionarily conserved in the indicated species. Alignment of the sequences around NFATc3 K384 is illustrated. g AsPC-1 cells expressing WT NFATc3 or NFATc3 K384R were cultured for 24 h under normoxia or hypoxia condition.

Article Snippet: Antibody against NFATc3 (A6666) was obtained from Abclonal.

Techniques: Immunoprecipitation, Western Blot, Transfection, Expressing, Cell Culture

Journal: Cell Death & Disease

Article Title: Hypoxia-induced NFATc3 deSUMOylation enhances pancreatic carcinoma progression

doi: 10.1038/s41419-022-04779-9

Figure Lengend Snippet: a, d, e, f, g Immunoprecipitation and immunoblot analyses were performed with the indicated antibodies. a AsPC-1 cells that stably expressed Flag-NFATc3 were transfected with non-specific siRNA (siCon) or SENPs siRNA for 72 h. b , c PANC-1 and AsPC-1 cells were cultured under normoxia or hypoxia condition for the indicated time. Immunoblot analyses ( b ) and RT-PCR ( c ) were performed (N.S. = not significant for the indicated comparison). d AsPC-1 cells that stably expressed Flag-NFATc3 were cultured under normoxia or hypoxia condition for 24 h. e PANC-1 and AsPC-1 cells were cultured under normoxia or hypoxia condition for 24 h. f AsPC-1 cells with or without SENP3 depletion and reconstituted expression of Flag-NFATc3 were cultured for 24 h under normoxia or hypoxia. g AsPC-1 cells that stably expressed Flag-NFATc3 were transfected with V5-WT SENP3 or V5-SENP3 C532A plasmids as indicated.

Article Snippet: Antibody against NFATc3 (A6666) was obtained from Abclonal.

Techniques: Immunoprecipitation, Western Blot, Stable Transfection, Transfection, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Comparison, Expressing

Journal: Cell Death & Disease

Article Title: Hypoxia-induced NFATc3 deSUMOylation enhances pancreatic carcinoma progression

doi: 10.1038/s41419-022-04779-9

Figure Lengend Snippet: a – c Cytosolic and nuclear fractions were prepared and immunoblotting analyses with the indicated antibodies were performed. a. PANC-1 and AsPC-1 cells that stably expressed Flag-WT NFATc3 or Flag-NATc3 K384R were cultured under normoxia or hypoxia condition for 24 h. b PANC-1 and AsPC-1 cells with or without the expression of SENP3 sgRNA expressed Flag-WT NFATc3 or Flag-NFATc3 K384R proteins. The indicated cells were cultured under normoxia or hypoxia condition for 24 h. c Parental and the indicated SENP3-knockout AsPC-1 and PANC-1 cells were cultured under normoxia or hypoxia condition for 24 h. d AsPC-1 cells with or without the expression of SENP3 sgRNA expressed Flag-WT NFATc3 or Flag-NFATc3 K384R proteins. Immunoprecipitation and immunoblot analyses were performed with the indicated antibodies. e ChIP assay with an anti-NFATc3 antibody and quantitative PCR with primers against the promoter regions of MYC in AsPC-1 cells with or without SENP3 depletion were performed. Two-sided t -test analyses were conducted. The data are presented as the means ± S.D. of three independent experiments ( n = 3). ** P < 0.01. f Flag-WT NFATc3 or Flag-NFATc3 K384R was expressed in AsPC-1 and PANC-1 cells with or without the expression of SENP3 sgRNA. The indicated cells were transfected with luciferase reporter plasmid (NFATc3-Luc) and stimulated with or without hypoxia for 12 h. The data are presented as the mean ± SD of triplicate samples (*** P < 0.001; N.S. = not significant for the indicated comparison).

Article Snippet: Antibody against NFATc3 (A6666) was obtained from Abclonal.

Techniques: Western Blot, Stable Transfection, Cell Culture, Expressing, Knock-Out, Immunoprecipitation, Real-time Polymerase Chain Reaction, Transfection, Luciferase, Plasmid Preparation, Comparison

Journal: Cell Death & Disease

Article Title: Hypoxia-induced NFATc3 deSUMOylation enhances pancreatic carcinoma progression

doi: 10.1038/s41419-022-04779-9

Figure Lengend Snippet: a , b Immunoblotting analyses with the indicated antibodies were performed. a AsPC-1 cells and PANC-1 cells with a vector expressing control sgCon or sgNFATc3 and with reconstituted expression of WT rNFATc3 or rNFATc3 K384R. b AsPC-1 cells and PANC-1 cells with depleted NFATc3 and reconstituted expression of WT rNFAc3 or rNFATc3 K384R were cultured with or without hypoxia for 24 h. c – f PANC-1 cells with depleted NFATc3 and reconstituted expression of WT rNFATc3 or rNFATc3 K384R were cultured with or without hypoxia. c, d Indicated PANC-1 cells were plated for the indicated periods under hypoxia before measuring cell proliferation ( c ) or for 2 weeks before counting colony numbers ( d ). Data are presented as the means ± SD from three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0.001. e Indicated PANC-1 cells were cultured in serum-free medium for 24 h under normoxia or hypoxia, and concentrations of the MMP2 in culture supernatants were measured by ELISA. Data are presented as the means ± SD from three independent experiments. *** p < 0.001. f The migration and invasion of the indicated PANC-1 cells were examined by the transwell assay. The membrane was photographed using a digital camera mounted onto a microscope. Scale bars, 50 μm. Data are presented as mean ± S.D. *** P < 0.001.

Article Snippet: Antibody against NFATc3 (A6666) was obtained from Abclonal.

Techniques: Western Blot, Plasmid Preparation, Expressing, Control, Cell Culture, Enzyme-linked Immunosorbent Assay, Migration, Transwell Assay, Membrane, Microscopy

Journal: Cell Death & Disease

Article Title: Hypoxia-induced NFATc3 deSUMOylation enhances pancreatic carcinoma progression

doi: 10.1038/s41419-022-04779-9

Figure Lengend Snippet: A total of 3 × 10 6 control or SENP3 depleted PANC-1 cells with NFATc3 depletion and reconstituted expression of the WT NFATc3 or rNFATc3 K384R were subcutaneously injected into the athymic nude mice. Representative tumor xenografts were shown ( n = 6 mice per group) ( a ). Tumor weights were calculated ( b ). Tumor growth was measured every other day beginning on day 6 and tumor volumes were calculated ( c ). Immunochemistry staining of the tumor sections were performed with antibodies against Ki67, c-Myc, and Flag. Representative images are shown ( d ). Scale bars, 10 μm. Data represent the means ± s.e.m. *** P < 0.001.

Article Snippet: Antibody against NFATc3 (A6666) was obtained from Abclonal.

Techniques: Control, Expressing, Injection, Staining

Journal: Cell Death & Disease

Article Title: Hypoxia-induced NFATc3 deSUMOylation enhances pancreatic carcinoma progression

doi: 10.1038/s41419-022-04779-9

Figure Lengend Snippet: Hypoxia resulted in SENP3-mediated deSUMOylation of NFATc3 at K384, suppressing the interaction between NFATc3 and its phosphokinase GSK-3β and subsequent increasing NFATc3 nuclear occupancy. In the nuclei of PDAC cells, NFATc3 formed complexes with other factors to coordinate target gene expression, which promoted PDAC progression.

Article Snippet: Antibody against NFATc3 (A6666) was obtained from Abclonal.

Techniques: Targeted Gene Expression