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nfatc3 (Proteintech)

Name: nfatc3
Brand: Proteintech
Rating: 93 (36 reviews)

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Proteintech nfatc3
Nfatc3, supplied by Proteintech, used in various techniques. Bioz Stars score: 93/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/nfatc3/product/Proteintech
Average 93 stars, based on 36 article reviews

nfatc3 - by Bioz Stars, 2026-02

93/100 stars

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a. Conserved miR-200 targeting sequence on the 3’UTR sequences of Csnk1a1 and Btrc . b & c. Luciferase reporter assay for Csnk1a1 (b) & Btrc (c) showing the functional relevance of miR-200 binding sites to their respective 3’UTR. A Student’s t-test was performed for statistical analysis. d. Spatial transcriptomics clusters of control and induced skin at P3. e. Schematics of scHolography analysis from ST and scRNA-seq together. f. Expression of selected marker genes along the epidermal-to-matrix axis. The solid line connects the median gene expression level at each computed distance bin along the epidermal-to-matrix axis. g-i. Expression of Wnt10a (g), Wnt10b (h) , and Sox9 (i) along the epidermal-to-matrix axis shows upregulation of Wnt10a and Wnt10b , as well as downregulation of Sox9 in the upper HF region. Solid lines connect the mean expression level for individual genes for each sample across the spatial bins. Wilcoxon test was performed for statistical analysis. j. Representative contour plot for HF-keratinocytes marked by EpCAM-APC + /SOX9-eGFP + from 2 control and 3 induced skin samples harvested at P1. k. Modal distribution of GFP intensity of the EpCAM + /SOX9-eGFP + of both control (red) and induced (blue) cells. l. SOX9 IF shows uHF-specific downregulation in induced HF compared to control. Scale bar: 20 μm. m. NFATc1 and PPARγ downregulation in induced HF reveals compromised SOX9 function. Scale bar: 20 μm. n. In SOX9 cKO and miR-200 induced samples, LEF1+ cells were detected in the uHF region marked by a white bracket whereas these LEF1+ cells were absent in the control uHF. White brackets annotate the uHF region. Scale bar: 20 μm.

Journal: bioRxiv

Article Title: Coordinated inhibition of SOX9 and cell cycle progression by microRNA-200 restricts sebaceous gland fate specification

doi: 10.64898/2026.01.09.698672

Figure Lengend Snippet: a. Conserved miR-200 targeting sequence on the 3’UTR sequences of Csnk1a1 and Btrc . b & c. Luciferase reporter assay for Csnk1a1 (b) & Btrc (c) showing the functional relevance of miR-200 binding sites to their respective 3’UTR. A Student’s t-test was performed for statistical analysis. d. Spatial transcriptomics clusters of control and induced skin at P3. e. Schematics of scHolography analysis from ST and scRNA-seq together. f. Expression of selected marker genes along the epidermal-to-matrix axis. The solid line connects the median gene expression level at each computed distance bin along the epidermal-to-matrix axis. g-i. Expression of Wnt10a (g), Wnt10b (h) , and Sox9 (i) along the epidermal-to-matrix axis shows upregulation of Wnt10a and Wnt10b , as well as downregulation of Sox9 in the upper HF region. Solid lines connect the mean expression level for individual genes for each sample across the spatial bins. Wilcoxon test was performed for statistical analysis. j. Representative contour plot for HF-keratinocytes marked by EpCAM-APC + /SOX9-eGFP + from 2 control and 3 induced skin samples harvested at P1. k. Modal distribution of GFP intensity of the EpCAM + /SOX9-eGFP + of both control (red) and induced (blue) cells. l. SOX9 IF shows uHF-specific downregulation in induced HF compared to control. Scale bar: 20 μm. m. NFATc1 and PPARγ downregulation in induced HF reveals compromised SOX9 function. Scale bar: 20 μm. n. In SOX9 cKO and miR-200 induced samples, LEF1+ cells were detected in the uHF region marked by a white bracket whereas these LEF1+ cells were absent in the control uHF. White brackets annotate the uHF region. Scale bar: 20 μm.

Article Snippet: Primary antibodies and dilutions used in this study are: PPARγ (1:400; Cell Signaling Technology #2443), SCD1 (1:400; Santa Cruz Biotechnology #sc-14719), LEF1 (1:400; Cell Signaling Technology #2230), FOXC1 (1:100; Cell Signaling Technology #8758), KRT5 (1:2000; Covance #SIG-3475), β4 integrin (1:400; clone 346-11A; BD Pharmingen #553745), SOX9 (1:200; Milipore Sigma #HPA001758), Alexa Fluor 488 conjugated with SOX9 (1:200; Millipore Sigma #AB5535-AF488), NFATc1 (1:10; Developmental Studies Hybridoma Bank #7A6).

Techniques: Sequencing, Luciferase, Reporter Assay, Functional Assay, Binding Assay, Control, Expressing, Marker, Gene Expression

Molecular mechanism by which miR-424/322 modulates the NFATc pathway. (A) miR-322 expression in AngII-induced H9c2 cells. (B) miR-424 expression in AngII-induced HCFs. (C) NFATc3 was bound to the promoter region of miR-322 and furin, as shown by ChIP assays using RT‒qPCR. (D) NFATc3 was bound to the promoter region of miR-424 and furin, as shown by luciferase assays in HEK293 cells. (E) miR-424 was bound to target mRNA as shown by luciferase assays in HEK293 cells. The data are expressed as the mean ± SD. ∗ p < 0.05.

Journal: Biomedical Journal

Article Title: miR-424/322 attenuates cardiac remodeling by modulating the nuclear factor-activated T-cell 3/furin pathway

doi: 10.1016/j.bj.2024.100818

Figure Lengend Snippet: Molecular mechanism by which miR-424/322 modulates the NFATc pathway. (A) miR-322 expression in AngII-induced H9c2 cells. (B) miR-424 expression in AngII-induced HCFs. (C) NFATc3 was bound to the promoter region of miR-322 and furin, as shown by ChIP assays using RT‒qPCR. (D) NFATc3 was bound to the promoter region of miR-424 and furin, as shown by luciferase assays in HEK293 cells. (E) miR-424 was bound to target mRNA as shown by luciferase assays in HEK293 cells. The data are expressed as the mean ± SD. ∗ p < 0.05.

Article Snippet: Membranes were probed with monoclonal antibodies against NFATc3 (Proteintech, 18222-1-AP), α-SMA (Abcam, ab5694), ANP (GeneTex, GTX109255), BNP (Santa Cruz, sc-271,185), furin (Abcam, ab183495), Col1A1 (Santa Cruz, sc-293,182), Smad2/3 (CST, #8828), GAPDH (Santa Cruz, sc-32233), and β-actin (Millipore, #MAB1501).

Techniques: Expressing, Luciferase

NFATc3 is upregulated during human and mouse AAD. (A) t -Distributed stochastic neighbor embedding ( t -SNE) visualization of cell types isolated from human ascending aortic wall tissue via scRNA-seq ( GSE155468 ). (B) Mean expression values scaled via mean-centering and transformed to a scale from −2 to 2. (C) Boxplot of NFATc3 expression level across major cell types. (D) Relative NFATc3 mRNA in the ascending aortas of patients undergoing coronary bypass, but without AAD (Con), and patients with TAAD. n = 36. (E) Relative NFATc3 protein levels in ascending aortas of patients with or without TAAD, and in abdominal aortas of patients with AAA ( n = 8). (F) Western blot analysis of NFATc3 levels in the cytoplasm and nucleus of aortas from patients without TAAD and those with TAAD or AAA ( n = 4). (G) NFATc3 and ACTA2 immunofluorescence in aortas of patients without TAAD or with TAAD or AAA ( n = 8). (H) Relative NFATc3 protein levels in mice aortas treated with saline, BAPN/AngII, or AAV- Pcsk9 DY /AngII ( n = 8). (I) NFATc3 and ACTA2 immunofluorescence in mice aortas treated with saline, BAPN/AngII, or AAV- Pcsk9 DY /AngII ( n = 6). Data are presented as mean ± SD. (C, D) Mann–Whitney U-test with the exact method; two-tailed P -values; (E–I) One-way ANOVA followed by Dunnett’s correction; adjusted P -values.

Journal: Acta Pharmaceutica Sinica. B

Article Title: Cytoplasmic and nuclear NFATc3 cooperatively contributes to vascular smooth muscle cell dysfunction and drives aortic aneurysm and dissection

doi: 10.1016/j.apsb.2025.05.016

Figure Lengend Snippet: NFATc3 is upregulated during human and mouse AAD. (A) t -Distributed stochastic neighbor embedding ( t -SNE) visualization of cell types isolated from human ascending aortic wall tissue via scRNA-seq ( GSE155468 ). (B) Mean expression values scaled via mean-centering and transformed to a scale from −2 to 2. (C) Boxplot of NFATc3 expression level across major cell types. (D) Relative NFATc3 mRNA in the ascending aortas of patients undergoing coronary bypass, but without AAD (Con), and patients with TAAD. n = 36. (E) Relative NFATc3 protein levels in ascending aortas of patients with or without TAAD, and in abdominal aortas of patients with AAA ( n = 8). (F) Western blot analysis of NFATc3 levels in the cytoplasm and nucleus of aortas from patients without TAAD and those with TAAD or AAA ( n = 4). (G) NFATc3 and ACTA2 immunofluorescence in aortas of patients without TAAD or with TAAD or AAA ( n = 8). (H) Relative NFATc3 protein levels in mice aortas treated with saline, BAPN/AngII, or AAV- Pcsk9 DY /AngII ( n = 8). (I) NFATc3 and ACTA2 immunofluorescence in mice aortas treated with saline, BAPN/AngII, or AAV- Pcsk9 DY /AngII ( n = 6). Data are presented as mean ± SD. (C, D) Mann–Whitney U-test with the exact method; two-tailed P -values; (E–I) One-way ANOVA followed by Dunnett’s correction; adjusted P -values.

Article Snippet: The supernatant was incubated with anti-NFATc3 antibody (18222-1-AP; 4 μg; ProteinTech) or control rabbit IgG antibody (#2729; 4 μg; Cell Signaling Technology) at 4 °C overnight.

Techniques: Isolation, Expressing, Transformation Assay, Western Blot, Immunofluorescence, Saline, MANN-WHITNEY, Two Tailed Test

VSMC–NFATc3 deficiency mitigates AAD in mice. (A–H) Five-week-old Nfatc3 fl/fl or Nfatc3 smcKO male mice administered BAPN (0.3 g/kg/day) for 28 days, followed by AngII infusion (1000 ng/kg/min) for 3 days ( n = 22). (A) Mouse aorta macrographs. (B) Thoracic aorta ultrasound images. (C) Survival curves analyzed via Kaplan–Meier and compared using log-rank tests. (D) AAD incidence per group. (E) Maximal aortic diameter ex vivo ( n = 15 for Nfatc3 fl/fl ; n = 22 for Nfatc3 smcKO ). (F) H&E, van Geison (elastin), and Masson staining of mouse ascending aorta. (G, H) Elastin degradation grade (G) and collagen content (H) in the aortic wall. (I–K) Eight-week-old Nfatc3 fl/fl or Nfatc3 smcKO mice were injected AAV- Pcsk9 DY via the tail vein and fed a Western-type diet. The mice were infused with AngII (1500 ng/kg/min) after 2 weeks for another 4 weeks. (I) Mouse aorta macrographs. (J) AAA incidence per group. (K) Maximal aortic diameter ex vivo ( n = 17 for Nfatc3 fl/fl ; n = 21 for Nfatc3 smcKO ). Mice that died from aortic rupture were not included in the measurements. Data are presented as mean ± SD. (E, H, K) Unpaired Student’s t -test; two-tailed P -values. (G) Mann–Whitney U-test with the exact method; two-tailed P -values.

Journal: Acta Pharmaceutica Sinica. B

Article Title: Cytoplasmic and nuclear NFATc3 cooperatively contributes to vascular smooth muscle cell dysfunction and drives aortic aneurysm and dissection

doi: 10.1016/j.apsb.2025.05.016

Figure Lengend Snippet: VSMC–NFATc3 deficiency mitigates AAD in mice. (A–H) Five-week-old Nfatc3 fl/fl or Nfatc3 smcKO male mice administered BAPN (0.3 g/kg/day) for 28 days, followed by AngII infusion (1000 ng/kg/min) for 3 days ( n = 22). (A) Mouse aorta macrographs. (B) Thoracic aorta ultrasound images. (C) Survival curves analyzed via Kaplan–Meier and compared using log-rank tests. (D) AAD incidence per group. (E) Maximal aortic diameter ex vivo ( n = 15 for Nfatc3 fl/fl ; n = 22 for Nfatc3 smcKO ). (F) H&E, van Geison (elastin), and Masson staining of mouse ascending aorta. (G, H) Elastin degradation grade (G) and collagen content (H) in the aortic wall. (I–K) Eight-week-old Nfatc3 fl/fl or Nfatc3 smcKO mice were injected AAV- Pcsk9 DY via the tail vein and fed a Western-type diet. The mice were infused with AngII (1500 ng/kg/min) after 2 weeks for another 4 weeks. (I) Mouse aorta macrographs. (J) AAA incidence per group. (K) Maximal aortic diameter ex vivo ( n = 17 for Nfatc3 fl/fl ; n = 21 for Nfatc3 smcKO ). Mice that died from aortic rupture were not included in the measurements. Data are presented as mean ± SD. (E, H, K) Unpaired Student’s t -test; two-tailed P -values. (G) Mann–Whitney U-test with the exact method; two-tailed P -values.

Techniques: Ex Vivo, Staining, Injection, Western Blot, Two Tailed Test, MANN-WHITNEY

VSMC–NFATc3 overexpression aggravates AAD in mice. (A–H) Five-week-old Nfatc3 -KI fl/fl or Nfatc3 smcKI male mice were administered BAPN (0.3 g/kg/day) for 28 d before infusion with AngII (1000 ng/kg/min) for 3 d ( n = 19 for Nfatc3 -KI fl/fl ; n = 20 for Nfatc3 smcKI ). (A) Mouse aorta macrographs. (B) Thoracic aorta ultrasound images. (C) Survival curves per group analyzed using Kaplan–Meier and compared using log-rank tests. (D) AAD incidence. (E) Maximal aortic diameter ex vivo ( n = 13 for Nfatc3 -KI fl/fl ; n = 7 for Nfatc3 smcKI ). (F) H&E, van Geison (elastin), and Masson staining of the mouse ascending aorta. (G, H) Elastin degradation grade (G) and collagen content (H) in the aortic wall. (I–K) Eight-week-old Nfatc3 -KI fl/fl or Nfatc3 smcKI mice injected AAV- Pcsk9 DY via tail vein and fed a Western-type diet. Two weeks later, the mice were infused with AngII (1500 ng/kg/min) for 4 weeks. (I) Mouse aorta macrographs. (J) AAA incidence. (K) Maximal aortic diameter ex vivo ( n = 13 for Nfatc3 -KI fl/fl ; n = 10 for Nfatc3 smcKI ). Mice that died from aortic rupture were not included in the measurements. Data are presented as mean ± SD. (E, H) Unpaired Student’s t -test; two-tailed P -values. (G, K) Mann–Whitney U-test with the exact method; two-tailed P -values.

Journal: Acta Pharmaceutica Sinica. B

Article Title: Cytoplasmic and nuclear NFATc3 cooperatively contributes to vascular smooth muscle cell dysfunction and drives aortic aneurysm and dissection

doi: 10.1016/j.apsb.2025.05.016

Figure Lengend Snippet: VSMC–NFATc3 overexpression aggravates AAD in mice. (A–H) Five-week-old Nfatc3 -KI fl/fl or Nfatc3 smcKI male mice were administered BAPN (0.3 g/kg/day) for 28 d before infusion with AngII (1000 ng/kg/min) for 3 d ( n = 19 for Nfatc3 -KI fl/fl ; n = 20 for Nfatc3 smcKI ). (A) Mouse aorta macrographs. (B) Thoracic aorta ultrasound images. (C) Survival curves per group analyzed using Kaplan–Meier and compared using log-rank tests. (D) AAD incidence. (E) Maximal aortic diameter ex vivo ( n = 13 for Nfatc3 -KI fl/fl ; n = 7 for Nfatc3 smcKI ). (F) H&E, van Geison (elastin), and Masson staining of the mouse ascending aorta. (G, H) Elastin degradation grade (G) and collagen content (H) in the aortic wall. (I–K) Eight-week-old Nfatc3 -KI fl/fl or Nfatc3 smcKI mice injected AAV- Pcsk9 DY via tail vein and fed a Western-type diet. Two weeks later, the mice were infused with AngII (1500 ng/kg/min) for 4 weeks. (I) Mouse aorta macrographs. (J) AAA incidence. (K) Maximal aortic diameter ex vivo ( n = 13 for Nfatc3 -KI fl/fl ; n = 10 for Nfatc3 smcKI ). Mice that died from aortic rupture were not included in the measurements. Data are presented as mean ± SD. (E, H) Unpaired Student’s t -test; two-tailed P -values. (G, K) Mann–Whitney U-test with the exact method; two-tailed P -values.

Techniques: Over Expression, Ex Vivo, Staining, Injection, Western Blot, Two Tailed Test, MANN-WHITNEY

VSMC–NFATc3 facilitates AAD progression in the BAPN model. (A) Relative NFATc3 mRNA levels in ascending aortas of patients without TAAD undergoing coronary bypass (Control, n = 36) and TAAD with and without hypertension ( n = 25; 11, respectively). (B) NFATc3 Western blot in mouse aortas with different AngII treatment durations ( n = 4). (C–I) Five-week-old male Nfatc3 fl/fl ( n = 15), Nfatc3 smcKO ( n = 15), Nfatc3 -KI fl/fl ( n = 17), and Nfatc3 smcKI ( n = 18) mice were administered BAPN (0.6 g/kg/day) for 28 days. (C) Aorta macrographs. (D) TAAD incidence. (E) Thoracic aorta ultrasound images. (F) The survival rate was estimated using Kaplan–Meier and compared using the log-rank test. (G) Maximum aortic diameter. (H) H&E, elastin, and Masson aorta staining. (I, J) Phenylephrine (PE)-induced vascular contraction (I) and acetylcholine (Ach)-induced endothelium-dependent vasodilation (J) of aortic rings from Nfatc3 fl/fl and Nfatc3 smcKO mice treated with vehicle or BAPN for 28 days ( n = 8). (K) Western blot and ACTA2, CNN1, SM22 α , and OPN quantification in aortas ( n = 6). Mice that died from aortic rupture were not included in the measurements. Data are presented as mean ± SD. (A, B, G) One-way ANOVA with Tukey’s correction; adjusted P -values. (I–K) Two-way ANOVA with Tukey’s correction; adjusted P -values. NS, no significance ( P > 0.05).

Journal: Acta Pharmaceutica Sinica. B

Article Title: Cytoplasmic and nuclear NFATc3 cooperatively contributes to vascular smooth muscle cell dysfunction and drives aortic aneurysm and dissection

doi: 10.1016/j.apsb.2025.05.016

Figure Lengend Snippet: VSMC–NFATc3 facilitates AAD progression in the BAPN model. (A) Relative NFATc3 mRNA levels in ascending aortas of patients without TAAD undergoing coronary bypass (Control, n = 36) and TAAD with and without hypertension ( n = 25; 11, respectively). (B) NFATc3 Western blot in mouse aortas with different AngII treatment durations ( n = 4). (C–I) Five-week-old male Nfatc3 fl/fl ( n = 15), Nfatc3 smcKO ( n = 15), Nfatc3 -KI fl/fl ( n = 17), and Nfatc3 smcKI ( n = 18) mice were administered BAPN (0.6 g/kg/day) for 28 days. (C) Aorta macrographs. (D) TAAD incidence. (E) Thoracic aorta ultrasound images. (F) The survival rate was estimated using Kaplan–Meier and compared using the log-rank test. (G) Maximum aortic diameter. (H) H&E, elastin, and Masson aorta staining. (I, J) Phenylephrine (PE)-induced vascular contraction (I) and acetylcholine (Ach)-induced endothelium-dependent vasodilation (J) of aortic rings from Nfatc3 fl/fl and Nfatc3 smcKO mice treated with vehicle or BAPN for 28 days ( n = 8). (K) Western blot and ACTA2, CNN1, SM22 α , and OPN quantification in aortas ( n = 6). Mice that died from aortic rupture were not included in the measurements. Data are presented as mean ± SD. (A, B, G) One-way ANOVA with Tukey’s correction; adjusted P -values. (I–K) Two-way ANOVA with Tukey’s correction; adjusted P -values. NS, no significance ( P > 0.05).

Techniques: Control, Western Blot, Staining

NFATc3 promotes ECM degradation by transcriptionally upregulating MMP9 and MMP2 expression in VSMCs. (A) Gene Ontology term enrichment analysis of the biological process influenced by NFATc3 deficiency based on the RNA-seq dataset. Downregulated processes in the aortas of Nfatc3 smcKO mice compared to those in Nfatc3 fl/fl mice treated with BAPN (0.6 g/kg/day) for 28 days ( n = 3). (B) Gene expression profiles were compared between Nfatc3 smcKO to Nfatc3 fl/fl mice treated with BAPN, and heatmaps were generated based on the expression of significantly different genes related to ECM organization (blue, downregulated; red, upregulated). Genes with a corresponding adjusted P < 0.05 were considered statistically significant. (C) Relative mRNA levels of genes related to ECM organization in the aortas of Nfatc3 smcKO and Nfatc3 fl/fl mice treated with BAPN ( n = 6). (D) MMP2 and MMP9 Western blot in the aortas of Nfatc3 smcKO and Nfatc3 fl/fl mice treated with vehicle or BAPN ( n = 6). (E) MMP2 and MMP9 protein levels in HASMCs infected with Ad- LacZ and Ad- NFATc3 ( n = 6). (F) Immunofluorescence of in situ zymography and immunostaining for ACTA2 in Nfatc3 fl/fl and Nfatc3 smcKO ascending aortas after vehicle or BAPN treatment ( n = 6). (G) Gelatin zymography for detecting pro-MMP9, active-MMP9, pro-MMP2, and active-MMP2 in the ascending aortas of Nfatc3 fl/fl and Nfatc3 smcKO mice after vehicle or BAPN treatment. (H, I) Correlation analysis of NFATc3 mRNA with MMP9 (H) or MMP2 (I) levels in the ascending aortas of patients without AAD (Con) ( n = 36) and with TAAD ( n = 36). (J) NFATc3-binding motifs. (K) Luciferase reporter analysis in 293T cells ( n = 6). (L) ChIP-qPCR validation of H3K27ac and H3K4me3 enrichment on the Mmp9 and Mmp2 promoter ( n = 6). Data are presented as mean ± SD. (C, E, L) Unpaired Student’s t -test; two-tailed P -values. (D, F, K) Two-way ANOVA with Tukey’s correction; adjusted P -values. (H, I) Spearman’s or Pearson’s correlation coefficient test; regression coefficients. NS, no significance ( P > 0.05).

Journal: Acta Pharmaceutica Sinica. B

Article Title: Cytoplasmic and nuclear NFATc3 cooperatively contributes to vascular smooth muscle cell dysfunction and drives aortic aneurysm and dissection

doi: 10.1016/j.apsb.2025.05.016

Figure Lengend Snippet: NFATc3 promotes ECM degradation by transcriptionally upregulating MMP9 and MMP2 expression in VSMCs. (A) Gene Ontology term enrichment analysis of the biological process influenced by NFATc3 deficiency based on the RNA-seq dataset. Downregulated processes in the aortas of Nfatc3 smcKO mice compared to those in Nfatc3 fl/fl mice treated with BAPN (0.6 g/kg/day) for 28 days ( n = 3). (B) Gene expression profiles were compared between Nfatc3 smcKO to Nfatc3 fl/fl mice treated with BAPN, and heatmaps were generated based on the expression of significantly different genes related to ECM organization (blue, downregulated; red, upregulated). Genes with a corresponding adjusted P < 0.05 were considered statistically significant. (C) Relative mRNA levels of genes related to ECM organization in the aortas of Nfatc3 smcKO and Nfatc3 fl/fl mice treated with BAPN ( n = 6). (D) MMP2 and MMP9 Western blot in the aortas of Nfatc3 smcKO and Nfatc3 fl/fl mice treated with vehicle or BAPN ( n = 6). (E) MMP2 and MMP9 protein levels in HASMCs infected with Ad- LacZ and Ad- NFATc3 ( n = 6). (F) Immunofluorescence of in situ zymography and immunostaining for ACTA2 in Nfatc3 fl/fl and Nfatc3 smcKO ascending aortas after vehicle or BAPN treatment ( n = 6). (G) Gelatin zymography for detecting pro-MMP9, active-MMP9, pro-MMP2, and active-MMP2 in the ascending aortas of Nfatc3 fl/fl and Nfatc3 smcKO mice after vehicle or BAPN treatment. (H, I) Correlation analysis of NFATc3 mRNA with MMP9 (H) or MMP2 (I) levels in the ascending aortas of patients without AAD (Con) ( n = 36) and with TAAD ( n = 36). (J) NFATc3-binding motifs. (K) Luciferase reporter analysis in 293T cells ( n = 6). (L) ChIP-qPCR validation of H3K27ac and H3K4me3 enrichment on the Mmp9 and Mmp2 promoter ( n = 6). Data are presented as mean ± SD. (C, E, L) Unpaired Student’s t -test; two-tailed P -values. (D, F, K) Two-way ANOVA with Tukey’s correction; adjusted P -values. (H, I) Spearman’s or Pearson’s correlation coefficient test; regression coefficients. NS, no significance ( P > 0.05).

Techniques: Expressing, RNA Sequencing, Gene Expression, Generated, Western Blot, Infection, Immunofluorescence, In Situ, Zymography, Immunostaining, Binding Assay, Luciferase, ChIP-qPCR, Biomarker Discovery, Two Tailed Test

Cytoplasmic NFATc3 facilitates VSMC contractile-to-synthetic phenotype switching by promoting global protein synthesis. (A) Representative images and quantification of NFATc3 and ACTA2 expression using immunofluorescence staining in MASMCs isolated from mice treated with vehicle and BAPN for 28 days ( n = 6). (B) NFATc3-associated protein subcellular localization based on LC–MS/MS. (C) Gene Ontology analysis of NFATc3-associated proteins identified using LC–MS/MS. (D) Polysome profiling shows NFATc3 distribution in MASMCs using sucrose density gradient centrifugation ( n = 3). Western blot of NFATc3 in the elution profiles. (E) Representative Western blot images and quantification of puromycin incorporation assays from MASMCs isolated from Nfatc3 -KI fl/fl and Nfatc3 smcKI mice ( n = 4). (F) HPG incorporation assay in VSMCs isolated from the aortas of Nfatc3 -KI fl/fl and Nfatc3 smcKI mice treated with BAPN for 28 days ( n = 4). (G) Western blot and quantification of ACTA2, CNN1, SM22 α , and OPN in MASMCs treated with PDGF-BB for 24 h before a 12-h CHX treatment ( n = 4). (H) Representative Western blot images and quantification of ACTA2, CNN1, SM22 α , and OPN in MASMCs isolated from aortas of Nfatc3 -KI fl/fl and Nfatc3 smcKI mice and treated with PDGF-BB (20 μg/L) for 24 h before a 12-h CHX (50 μmol/L) treatment ( n = 4). (I) Co-immunoprecipitation of MASMC lysates with anti-NFATc3 antibodies. Western blot of RPL28 and RPS6 levels. Data are presented as mean ± SD. (E) Unpaired Student’s t -test; two-tailed P -values. (G) Two-way ANOVA with Tukey’s correction; adjusted P -values. (H) One-way ANOVA with Tukey’s correction; adjusted P -values.

Journal: Acta Pharmaceutica Sinica. B

Article Title: Cytoplasmic and nuclear NFATc3 cooperatively contributes to vascular smooth muscle cell dysfunction and drives aortic aneurysm and dissection

doi: 10.1016/j.apsb.2025.05.016

Figure Lengend Snippet: Cytoplasmic NFATc3 facilitates VSMC contractile-to-synthetic phenotype switching by promoting global protein synthesis. (A) Representative images and quantification of NFATc3 and ACTA2 expression using immunofluorescence staining in MASMCs isolated from mice treated with vehicle and BAPN for 28 days ( n = 6). (B) NFATc3-associated protein subcellular localization based on LC–MS/MS. (C) Gene Ontology analysis of NFATc3-associated proteins identified using LC–MS/MS. (D) Polysome profiling shows NFATc3 distribution in MASMCs using sucrose density gradient centrifugation ( n = 3). Western blot of NFATc3 in the elution profiles. (E) Representative Western blot images and quantification of puromycin incorporation assays from MASMCs isolated from Nfatc3 -KI fl/fl and Nfatc3 smcKI mice ( n = 4). (F) HPG incorporation assay in VSMCs isolated from the aortas of Nfatc3 -KI fl/fl and Nfatc3 smcKI mice treated with BAPN for 28 days ( n = 4). (G) Western blot and quantification of ACTA2, CNN1, SM22 α , and OPN in MASMCs treated with PDGF-BB for 24 h before a 12-h CHX treatment ( n = 4). (H) Representative Western blot images and quantification of ACTA2, CNN1, SM22 α , and OPN in MASMCs isolated from aortas of Nfatc3 -KI fl/fl and Nfatc3 smcKI mice and treated with PDGF-BB (20 μg/L) for 24 h before a 12-h CHX (50 μmol/L) treatment ( n = 4). (I) Co-immunoprecipitation of MASMC lysates with anti-NFATc3 antibodies. Western blot of RPL28 and RPS6 levels. Data are presented as mean ± SD. (E) Unpaired Student’s t -test; two-tailed P -values. (G) Two-way ANOVA with Tukey’s correction; adjusted P -values. (H) One-way ANOVA with Tukey’s correction; adjusted P -values.

Techniques: Expressing, Immunofluorescence, Staining, Isolation, Liquid Chromatography with Mass Spectroscopy, Gradient Centrifugation, Western Blot, Immunoprecipitation, Two Tailed Test

NFATc3 facilitates global protein synthesis by suppressing eEF2 phosphorylation. (A) Co-immunoprecipitation of MASMC lysates with anti-NFATc3 antibodies. The eEF2 level was determined using Western blot. (B) HEK293T cells were co-transfected with His-eEF2 and Flag-NFATc3 plasmids. Cell lysates were immunoprecipitated with anti-Flag antibodies, and the precipitates were analyzed using immunoblotting with anti-His antibodies. (C) Immunofluorescence for eEF2 and NFATc3 detection in MASMCs. (D) Nfatc3 fl/fl and Nfatc3 smcKO mice were treated with or without BAPN for 28 days. Western blot and quantification of p-eEF2 and eEF2 in aortic tissues ( n = 4). (E) Western blots of binding assays show the binding preference of NFATc3. HEK293T cells were transfected with eEF2-T56D and eEF2-T56A plasmids for 48 h, and lysates were immunoprecipitated with anti-NFATc3 antibodies ( n = 4). (F) Schematic illustration of NFATc3 structures and the presence/absence of binding between NFATc3 and eEF2 are indicated by + or – (top panel). Full-length His-eEF2 and various truncated Flag-NFATc3 forms were co-expressed in 293T cells, and immunoprecipitation with anti-Flag antibodies followed by Western blot (bottom panel) was performed. (G) Western blots and quantification of p-eEF2 and eEF2 in MASMCs transfected with vector, Plasmid-Nfatc3, or Plasmid-Nfatc3-Mutant before a 24-h PDGF-BB treatment ( n = 4). (H) Representative images and quantification of eEF2 and ACTA2, p-eEF2, and ACTA2 expression using immunofluorescence staining in the ascending aortas of patients without TAAD and patients with TAAD ( n = 8). (I) Relative eEF2 and p-eEF2 protein levels in the aortas of mice administered BAPN/AngII and AAV- Pcsk9 DY /AngII ( n = 6). (J) Western blot of puromycin incorporation assays and p-eEF2/eEF2 levels from MASMCs isolated from Nfatc3 fl/fl and Nfatc3 smcKO mice and treated with A484954 (10 μmol/L, 10 min) ( n = 4). Data are presented as mean ± SD. (D) Two-way ANOVA with Tukey’s correction; adjusted P -values. (E, H) Unpaired Student’s t -test; two-tailed P -values. (G, I, J) One-way ANOVA followed by Tukey’s correction; adjusted P -values. NS, no significance ( P > 0.05).

Journal: Acta Pharmaceutica Sinica. B

Article Title: Cytoplasmic and nuclear NFATc3 cooperatively contributes to vascular smooth muscle cell dysfunction and drives aortic aneurysm and dissection

doi: 10.1016/j.apsb.2025.05.016

Figure Lengend Snippet: NFATc3 facilitates global protein synthesis by suppressing eEF2 phosphorylation. (A) Co-immunoprecipitation of MASMC lysates with anti-NFATc3 antibodies. The eEF2 level was determined using Western blot. (B) HEK293T cells were co-transfected with His-eEF2 and Flag-NFATc3 plasmids. Cell lysates were immunoprecipitated with anti-Flag antibodies, and the precipitates were analyzed using immunoblotting with anti-His antibodies. (C) Immunofluorescence for eEF2 and NFATc3 detection in MASMCs. (D) Nfatc3 fl/fl and Nfatc3 smcKO mice were treated with or without BAPN for 28 days. Western blot and quantification of p-eEF2 and eEF2 in aortic tissues ( n = 4). (E) Western blots of binding assays show the binding preference of NFATc3. HEK293T cells were transfected with eEF2-T56D and eEF2-T56A plasmids for 48 h, and lysates were immunoprecipitated with anti-NFATc3 antibodies ( n = 4). (F) Schematic illustration of NFATc3 structures and the presence/absence of binding between NFATc3 and eEF2 are indicated by + or – (top panel). Full-length His-eEF2 and various truncated Flag-NFATc3 forms were co-expressed in 293T cells, and immunoprecipitation with anti-Flag antibodies followed by Western blot (bottom panel) was performed. (G) Western blots and quantification of p-eEF2 and eEF2 in MASMCs transfected with vector, Plasmid-Nfatc3, or Plasmid-Nfatc3-Mutant before a 24-h PDGF-BB treatment ( n = 4). (H) Representative images and quantification of eEF2 and ACTA2, p-eEF2, and ACTA2 expression using immunofluorescence staining in the ascending aortas of patients without TAAD and patients with TAAD ( n = 8). (I) Relative eEF2 and p-eEF2 protein levels in the aortas of mice administered BAPN/AngII and AAV- Pcsk9 DY /AngII ( n = 6). (J) Western blot of puromycin incorporation assays and p-eEF2/eEF2 levels from MASMCs isolated from Nfatc3 fl/fl and Nfatc3 smcKO mice and treated with A484954 (10 μmol/L, 10 min) ( n = 4). Data are presented as mean ± SD. (D) Two-way ANOVA with Tukey’s correction; adjusted P -values. (E, H) Unpaired Student’s t -test; two-tailed P -values. (G, I, J) One-way ANOVA followed by Tukey’s correction; adjusted P -values. NS, no significance ( P > 0.05).

Techniques: Phospho-proteomics, Immunoprecipitation, Western Blot, Transfection, Immunofluorescence, Binding Assay, Plasmid Preparation, Mutagenesis, Expressing, Staining, Isolation, Two Tailed Test

Supplementation with the eEF2 inhibitor cabamiquine mitigates the deleterious effects of NFATc3 overexpression on AAD development. (A) BAPN model establishment. (B–D) Four-week-old male Nfatc3 smcKO mice were treated with Lenti-Con or Lenti eEF2; 7 days after transfection, five-week-old mice were treated with vehicle or BAPN (0.6 g/kg/day) for 28 days ( n = 13). (B) aorta macrographs. (C) AAD incidence. (D)The survival rate was estimated using Kaplan–Meier and compared using the log-rank test. (E–K) Five-week-old male Nfatc3 smcKI mice were treated with BAPN (0.6 g/kg/day) for 28 days. Saline and cabamiquine (3 mg/kg/day) were administered orally for 28 days ( n = 15). (E) Aorta macrographs. (F) AAD incidence. (G) The survival rate was estimated using Kaplan–Meier and compared using the log-rank test. (H) Maximum aortic diameter. (I) H&E, van Geison (elastin), and Masson aorta staining. (J, K) Elastin degradation grade (J) and collagen content (K) in the aortic wall. (H–K) n = 5 for saline, n = 11 for cabamiquine. (L–O) Five-week-old Nfatc3 smcKI male mice were administered BAPN (0.3 g/kg/day) for 28 days before being infused with AngII (1000 ng/kg/min) for 3 days; saline and cabamiquine (3 mg/kg/day) were administered orally for 31 days ( n = 17 for saline, n = 18 for cabamiquine). (L) Aorta macrographs. (M) AAD incidence. (N) The survival rate was estimated using Kaplan–Meier and compared using the log-rank test. (O) Maximum aortic diameter (saline: n = 6; cabamiquine: n = 12). Mice that died from aortic rupture were not included in the measurements. Data are presented as mean ± SD. (H, K, O) Unpaired Student’s t -test; two-tailed P -values. (J) Mann–Whitney U -test with the exact method; two-tailed P -values.

Journal: Acta Pharmaceutica Sinica. B

Article Title: Cytoplasmic and nuclear NFATc3 cooperatively contributes to vascular smooth muscle cell dysfunction and drives aortic aneurysm and dissection

doi: 10.1016/j.apsb.2025.05.016

Figure Lengend Snippet: Supplementation with the eEF2 inhibitor cabamiquine mitigates the deleterious effects of NFATc3 overexpression on AAD development. (A) BAPN model establishment. (B–D) Four-week-old male Nfatc3 smcKO mice were treated with Lenti-Con or Lenti eEF2; 7 days after transfection, five-week-old mice were treated with vehicle or BAPN (0.6 g/kg/day) for 28 days ( n = 13). (B) aorta macrographs. (C) AAD incidence. (D)The survival rate was estimated using Kaplan–Meier and compared using the log-rank test. (E–K) Five-week-old male Nfatc3 smcKI mice were treated with BAPN (0.6 g/kg/day) for 28 days. Saline and cabamiquine (3 mg/kg/day) were administered orally for 28 days ( n = 15). (E) Aorta macrographs. (F) AAD incidence. (G) The survival rate was estimated using Kaplan–Meier and compared using the log-rank test. (H) Maximum aortic diameter. (I) H&E, van Geison (elastin), and Masson aorta staining. (J, K) Elastin degradation grade (J) and collagen content (K) in the aortic wall. (H–K) n = 5 for saline, n = 11 for cabamiquine. (L–O) Five-week-old Nfatc3 smcKI male mice were administered BAPN (0.3 g/kg/day) for 28 days before being infused with AngII (1000 ng/kg/min) for 3 days; saline and cabamiquine (3 mg/kg/day) were administered orally for 31 days ( n = 17 for saline, n = 18 for cabamiquine). (L) Aorta macrographs. (M) AAD incidence. (N) The survival rate was estimated using Kaplan–Meier and compared using the log-rank test. (O) Maximum aortic diameter (saline: n = 6; cabamiquine: n = 12). Mice that died from aortic rupture were not included in the measurements. Data are presented as mean ± SD. (H, K, O) Unpaired Student’s t -test; two-tailed P -values. (J) Mann–Whitney U -test with the exact method; two-tailed P -values.

Techniques: Over Expression, Transfection, Saline, Staining, Two Tailed Test, MANN-WHITNEY

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