anti nfat4  (R&D Systems)

Journal: Aging and Disease

Article Title: NFATc3 Promotes Pulmonary Inflammation and Fibrosis by Regulating Production of CCL2 and CXCL2 in Macrophages

doi: 10.14336/AD.2022.1202

Figure Lengend Snippet: NFATc3 expression is elevated in alveolar and interstitial macrophages of bleomycin treated IPF mice and IPF patients . NFATc3 +/+ (WT) mice were treated with BLM (1.4 U/kg, i.t) and lung tissue were analyzed on day 0, 3, 7 and 21. ( A ) The expression levels of NFATc1, NFATc2, NFATc3, NFATc4 and NFATc5 measured by real time RT-PCR. ( B ) The protein level of NFATc3 was analyzed by western blotting. ( C ) Arbitrary densitometry units of NFATc3 normalized to β-actin calculated using Image J software. ( D ) Immunohistochemical staining of NFATc3 in lung tissue from IPF or control mice (original magnification ×400, scale bar 100μm). ( E ) Immunohistochemical staining of NFATc3 in lung tissue from healthy donor or pulmonary fibrosis patients (original magnification ×400, scale bar 100μm). Data are shown as mean ± SEM. N =6 for each group, *p < 0.05, **p < 0.01, ***p < 0.001 (Saline vs 7, 21 days of BLM).

Article Snippet: For immunohistochemical staining, left lung sections from murine IPF models and IPF patients (around 2CM*2CM) were incubated at 4°C overnight with anti-NFATc3 antibody (4998, Cell Signaling, USA).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Software, Immunohistochemical staining, Staining, Control, Saline

Journal: Aging and Disease

Article Title: NFATc3 Promotes Pulmonary Inflammation and Fibrosis by Regulating Production of CCL2 and CXCL2 in Macrophages

doi: 10.14336/AD.2022.1202

Figure Lengend Snippet: NFATc3 expression is elevated in alveolar and interstitial macrophages of IPF patients and mice . ( A ) NFATc3 gene expression by microarray analysis of alveolar macrophage (AM) mRNA in healthy control subjects (N=45) and sporadic IPF patients (N=15). NFATc3 +/+ mice were treated with BLM (i.t) to induce pulmonary fibrosis, and after 3, 7 and 21 days, NFATc3 expression in (B) AMs and (C) IMs was detected by RT-qPCR analysis (N=6 for each group). ( D, E ) NFATc3 nuclear translocation in AMs and IMs was detected by immunofluorescence staining (scale bar 50μm). Data are shown as mean ± SEM. N =6 for each group, *p < 0.05, **p < 0.01, ***p < 0.001 (Saline vs 3, 7, 21 days of BLM).

Article Snippet: For immunohistochemical staining, left lung sections from murine IPF models and IPF patients (around 2CM*2CM) were incubated at 4°C overnight with anti-NFATc3 antibody (4998, Cell Signaling, USA).

Techniques: Expressing, Gene Expression, Microarray, Control, Quantitative RT-PCR, Translocation Assay, Immunofluorescence, Staining, Saline

Journal: Aging and Disease

Article Title: NFATc3 Promotes Pulmonary Inflammation and Fibrosis by Regulating Production of CCL2 and CXCL2 in Macrophages

doi: 10.14336/AD.2022.1202

Figure Lengend Snippet: NFATc3 deficiency alleviated bleomycin-induced pulmonary fibrosis in mice . NFATc3 +/+ and NFATc3 +/- mice were treated with bleomycin (1.4 U/kg, i.t) to establish pulmonary fibrosis. Sham control mice received the same volume of saline (i.t). After 21 days, lung tissue sections were stained by (A) hematoxylin-eosin (HE) and (B) Masson trichrome staining (original magnification ×400, scale bar100μm). ( C ) Severity of Pulmonary fibrosis among different experimental groups was compared by Ashcroft score. ( D, E ) Total RNA was extracted from lung tissues and the expression levels of ɑ-SMA and fibronectin were detected by RT-q-PCR. ( F ) Hydroxyproline was measured in different groups of mice. ( G, H ) The protein levels of ɑ-SMA were analyzed by western blotting and quantified using Image J software. Data are shown as mean ± SEM. N =6 for each group, *p < 0.05, **p < 0.01, ***p < 0.001 (NFATc3 +/+ vs NFATc3 +/- ).

Article Snippet: For immunohistochemical staining, left lung sections from murine IPF models and IPF patients (around 2CM*2CM) were incubated at 4°C overnight with anti-NFATc3 antibody (4998, Cell Signaling, USA).

Techniques: Control, Saline, Staining, Expressing, Western Blot, Software

Journal: Aging and Disease

Article Title: NFATc3 Promotes Pulmonary Inflammation and Fibrosis by Regulating Production of CCL2 and CXCL2 in Macrophages

doi: 10.14336/AD.2022.1202

Figure Lengend Snippet: NFATc3 deficiency decreased BLM-induced pulmonary inflammation in mice . NFATc3 +/+ and NFATc3 +/- mice were treated with normal saline or bleomycin (i.t) for 3 and 7 days. ( A, B ) lung tissue sections were stained by hematoxylin-eosin (HE) (scale bar 100μm). ( C ) Immune cell infiltration in to BALF was determined by counting total cells (D) Alveolar damage and protein leak in to BALF was determined by quantitating total BALF protein. Data are shown as mean ± SEM. N =6 for each group, *p < 0.05, **p < 0.01, ***p < 0.001 (NFATc3 +/+ vs NFATc3 +/- ).

Article Snippet: For immunohistochemical staining, left lung sections from murine IPF models and IPF patients (around 2CM*2CM) were incubated at 4°C overnight with anti-NFATc3 antibody (4998, Cell Signaling, USA).

Techniques: Saline, Staining

Journal: Aging and Disease

Article Title: NFATc3 Promotes Pulmonary Inflammation and Fibrosis by Regulating Production of CCL2 and CXCL2 in Macrophages

doi: 10.14336/AD.2022.1202

Figure Lengend Snippet: NFATc3 expression in pulmonary macrophages enhances susceptibility to BLM-induced IPF in mice . ( A ) Schematic diagram for the adoptive transfer of macrophages described in Materials and Methods. NFATc3 +/+ or NFATc3 +/- mice were adoptively transferred with NFATc3 +/+ or NFATc3 +/- BMDMs, and then treated with BLM (i.t) for 21 days. Lung sections were stained with (B) H&E to detect overall histological changes, and (C) Masson’s trichrome staining to detect collagen deposition (original magnification ×400, scale bar 100μm). ( D ) Severity of fibrosis was expressed as a means of individual Ashcroft scores of different experimental animals. ( E, F ) RNA was extracted from individual lung tissues and the mRNA levels of ɑ-SMA and fibronectin were detected by RT-qPCR. ( G, H ) The protein level of ɑ-SMA was analyzed by western blotting and quantified using Image J software. ( I ) Hydroxyproline was measured using hydroxyproline assay kit in different groups of mice. Data are shown as mean ± SEM. N =6 for each group, *p < 0.05, **p < 0.01, ***p < 0.001 (NFATc3 +/+ Macrophages→ NFATc3 +/+ / NFATc3 +/- mice vs NFATc3 +/- Macrophages→ NFATc3 +/+ / NFATc3 +/- mice).

Article Snippet: For immunohistochemical staining, left lung sections from murine IPF models and IPF patients (around 2CM*2CM) were incubated at 4°C overnight with anti-NFATc3 antibody (4998, Cell Signaling, USA).

Techniques: Expressing, Adoptive Transfer Assay, Staining, Quantitative RT-PCR, Western Blot, Software, Hydroxyproline Assay

Journal: Aging and Disease

Article Title: NFATc3 Promotes Pulmonary Inflammation and Fibrosis by Regulating Production of CCL2 and CXCL2 in Macrophages

doi: 10.14336/AD.2022.1202

Figure Lengend Snippet: NFATc3 regulates mRNA expression of CCL2 and CXCL2 in macrophages . MLE12 cells were cultured and stimulated with different concentrations of bleomycin, 0.05 nM, 0.5 nM and 5 nM for 24 hours, respectively. NFATc3 +/+ and NFATc3 +/- BMDMs were stimulated with different concentration of bleomycin-treated MLE12 culture supernatant (indicated as Med1, Med2 and Med3) for 24 hours, and then total RNA was extracted. The mRNA levels of (A) TNFα (B) IL-1β (C) IL-12P35 (D) IL-12P40 (E) IL-13 (F) TGF-β (G) CCL2 and (H) CXCL2 were measured by RT-qPCR. BMDMs from NFATc3 +/+ and NFATc3 +/- mice were treated separately with IL-33 (10 ng/ml) for 24, and the mRNA levels of (I) TNFα (J) IL-1β (K) IL-12P35 (L) IL-12P40 (M) IL-13 (N) TGF-β (O) CCL2 and (P) CXCL2 were measured by RT-qPCR. ( Q, R ) BMDMs from NFATc3 +/+ and NFATc3 +/- mice treated with Th2 cytokine mix (IL-4 and IL-13, 10 ng/mL each for 24 h) were analyzed for CCL2 and CXCL2 expression. Each experiment was independently repeated in triplicate, with duplicated wells. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 (NFATc3 +/+ vs NFATc3 +/- ).

Article Snippet: For immunohistochemical staining, left lung sections from murine IPF models and IPF patients (around 2CM*2CM) were incubated at 4°C overnight with anti-NFATc3 antibody (4998, Cell Signaling, USA).

Techniques: Expressing, Cell Culture, Concentration Assay, Quantitative RT-PCR

Journal: Aging and Disease

Article Title: NFATc3 Promotes Pulmonary Inflammation and Fibrosis by Regulating Production of CCL2 and CXCL2 in Macrophages

doi: 10.14336/AD.2022.1202

Figure Lengend Snippet: NFATc3 regulates protein levels of CCL2 and CXCL2 in macrophages . ( A, B ) NFATc3 +/+ and NFATc3 +/- BMDMs were stimulated with different concentrations of bleomycin-treated MLE12 culture supernatant (indicated as Med1, Med2 and Med3) for 24 h, and the protein levels of CCL2 and CXCL2 were detected using ELISA. ( C, D ) BMDMs from NFATc3 +/+ and NFATc3 +/- mice were treated separately with IL-33 (10 ng/ml) for 24, and the protein levels of CCL2 and CXCL2 were detected by ELISA. ( E, F ) BMDMs from NFATc3 +/+ and NFATc3 +/- mice treated with Th2 cytokine mix (IL-4 and IL-13,10 ng/mL each for 24 h) were analyzed for CCL2 and CXCL2 expression. Each experiment was independently repeated in triplicate, with duplicated wells. Data are shown as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 (NFATc3 +/+ vs NFATc3 +/- ).

Article Snippet: For immunohistochemical staining, left lung sections from murine IPF models and IPF patients (around 2CM*2CM) were incubated at 4°C overnight with anti-NFATc3 antibody (4998, Cell Signaling, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Expressing

Journal: Aging and Disease

Article Title: NFATc3 Promotes Pulmonary Inflammation and Fibrosis by Regulating Production of CCL2 and CXCL2 in Macrophages

doi: 10.14336/AD.2022.1202

Figure Lengend Snippet: NFATc3 promoted CCL2 and CXCL2 production in vivo . ( A, B ) The levels of the cytokines CCL2 and CXCL2 were detected by RT-qPCR in lungs from NFATc3 +/+ and NFATc3 +/- mice that were treated with saline or BLM (i.t) for 3, 7, and 21 days. ( C, D ) The protein levels of CCL2 and CXCL2 were detected by ELISA in lungs from NFATc3 +/+ and NFATc3 +/- mice that were treated with saline or BLM (i.t) for 3, 7, and 21 days. ( E, F ) The levels of the cytokines CCL2 and CXCL2 were detected by RT-qPCR in lungs from recipient mice that were adoptively transferred with NFATc3 deficient (NFATc3 +/- to NFATc3 +/+ ; NFATc3 +/- to NFATc3 +/- ) or NFATc3 sufficient macrophages (NFATc3+/+ to NFATc3 +/- ; NFATc3 +/+ to NFATc3 +/+ ). ( G, H ) The protein levels of CCL2 and CXCL2 were detected by ELISA in the lungs from recipient mice that were adoptively transferred with NFATc3 deficient (NFATc3 +/- ) or sufficient (NFATc3 +/+ ) macrophages. Data are shown as mean ± SEM. N=6 for each group, *p < 0.05, **p < 0.01, ***p < 0.001 (NFATc3 +/+ Macrophages→ NFATc3 +/+ / NFATc3 +/- mice vs NFATc3 +/- Macrophages→ NFATc3 +/+ / NFATc3 +/- mice).

Article Snippet: For immunohistochemical staining, left lung sections from murine IPF models and IPF patients (around 2CM*2CM) were incubated at 4°C overnight with anti-NFATc3 antibody (4998, Cell Signaling, USA).

Techniques: In Vivo, Quantitative RT-PCR, Saline, Enzyme-linked Immunosorbent Assay

Journal: Aging and Disease

Article Title: NFATc3 Promotes Pulmonary Inflammation and Fibrosis by Regulating Production of CCL2 and CXCL2 in Macrophages

doi: 10.14336/AD.2022.1202

Figure Lengend Snippet: CXCL2 restores BLM-induced pulmonary fibrosis in NFATc3 +/- mice . (A) Predicted binding sites of NFATc3 in the CXCL2 promoter analyzed using the JASPAR database. ( B ) Quantification of luciferase reporter activity of CXCL2 in Raw264.7 cells transfected with control and NFATc3 plasmids. ( C ) Schematic diagram of the experimental procedure. NFATc3 +/- mice were administrated with BLM (i.t) on day 1 and with rmCXCL2 (500ng, i.t) on day 3. Pulmonary fibrosis markers were measured on day 21. ( D ) H&E staining of lung sections (E) Masson trichrome staining to determine collagen deposition (original magnification ×400, scale bar 100μm). ( F ) Comparison of the Ashcroft score among the experimental groups. ( G, H ) The mRNA expression of α-SMA and fibronectin was measured by RT-qPCR. Data are shown as mean ± SEM. ( I, J ) The protein level of ɑ-SMA was analyzed by western blotting quantified using Image J software. ( K ) Hydroxyproline levels in different groups of experimental mice. N=6 for each group, (B) is *p <0.05 (CXCL2-pGL3+NFATc3-pDON223 vs CXCL2-pGL3+pDON223); (F-H, J, K) *p < 0.05, **p < 0.01 (Saline vs BLM and BLM+CXCL2).

Article Snippet: For immunohistochemical staining, left lung sections from murine IPF models and IPF patients (around 2CM*2CM) were incubated at 4°C overnight with anti-NFATc3 antibody (4998, Cell Signaling, USA).

Techniques: Binding Assay, Luciferase, Activity Assay, Transfection, Control, Staining, Comparison, Expressing, Quantitative RT-PCR, Western Blot, Software, Saline