Journal: iScience

Article Title: Estrogen upregulates NR4A1 to counter TGF beta induced pulmonary fibrosis therapeutic insights for IPF

doi: 10.1016/j.isci.2026.114756

Figure Lengend Snippet: Abnormal expression of NR4A1, p-NR4A1, and ER in lung tissues of patients with IPF (A–C) qPCR analysis of NR4A1, ERα, and ERβ mRNA expression in lung tissues from healthy controls (Ctrl, n = 11) and patients with IPF ( n = 23). (D) Representative immunoblotting images showing protein levels of NR4A1, p-NR4A1, ERα, and ERβ in lung tissues from Ctrl and IPF groups. β-Actin served as a loading control. (E) Quantification of immunoblotting results for NR4A1, p-NR4A1, p-NR4A1/NR4A1 ratio, ERα, and ERβ. (F–I) Correlation analysis between NR4A1 and ERα (F), NR4A1 and ERβ (G), p-NR4A1 and ERα (H), and p-NR4A1 and ERβ (I) in IPF lung tissues. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; Student’s t test for group comparisons, Pearson’s correlation for association analysis.

Article Snippet: Animal studies utilized male Nr4a1 knockout (NR4A1 −/− ) mice on a C57BL/6 background (Catalog S-KO-02468, Cyagen Biosciences) and 8-week-old male wild-type C57BL/6NCrl mice ((certificate No. HPPH-2022XS241, Beijing Vital River Laboratory Animal Technology Co., Ltd.).

Techniques: Expressing, Western Blot, Control

Journal: iScience

Article Title: Estrogen upregulates NR4A1 to counter TGF beta induced pulmonary fibrosis therapeutic insights for IPF

doi: 10.1016/j.isci.2026.114756

Figure Lengend Snippet: Silencing of NR4A1 in cells exacerbates TGF-β-induced fibrosis and increases the expression of α-SMA, SMAD2, fibronectin, and COL1A1 (A and B) qPCR and Western Blot analyses confirm reduced mRNA and protein expression levels of NR4A1 in MRC5 and HFL1 cells following siRNA-mediated NR4A1 silencing. (C and D) qPCR analysis of α-SMA, SMAD2, Fibronectin, and COL1A1 expression in NR4A1-silenced cells after TGF-β stimulation, showing elevated levels indicative of enhanced fibrosis. (E and F) Western blot analysis further confirms increased expression of α-SMA, SMAD2, fibronectin, and COL1A1 in NR4A1-silenced cells upon TGF-β stimulation, demonstrating that NR4A1 downregulation aggravates fibrotic responses. Each group n = 6; ∗ p < 0.05, ∗∗ p < 0.01 compared to the two groups.

Article Snippet: Animal studies utilized male Nr4a1 knockout (NR4A1 −/− ) mice on a C57BL/6 background (Catalog S-KO-02468, Cyagen Biosciences) and 8-week-old male wild-type C57BL/6NCrl mice ((certificate No. HPPH-2022XS241, Beijing Vital River Laboratory Animal Technology Co., Ltd.).

Techniques: Expressing, Western Blot

Journal: iScience

Article Title: Estrogen upregulates NR4A1 to counter TGF beta induced pulmonary fibrosis therapeutic insights for IPF

doi: 10.1016/j.isci.2026.114756

Figure Lengend Snippet: Increasing NR4A1 expression in cells attenuates TGF-β-induced fibrosis and decreases α-SMA, SMAD2, Fibronectin, and COL1A1 expression (A and B) NR4A1 overexpression in MRC5 and HFL1 cells induced by tetracycline, confirmed by increased NR4A1 mRNA and protein levels through qPCR and Western blot analyses. (C–J) qPCR analysis shows reduced expression levels of fibrosis markers α-SMA, SMAD2, Fibronectin, and COL1A1 in NR4A1-overexpressing cells following TGF-β stimulation, indicating diminished fibrotic responses. (K and L) Western blot analysis further confirms decreased protein levels of α-SMA, SMAD2, Fibronectin, and COL1A1 in TGF-β-stimulated NR4A1-overexpressing cells, demonstrating the antifibrotic effect of NR4A1 upregulation. Each group n = 6; ∗ p < 0.05, ∗∗ p < 0.01 compared to the two groups.

Article Snippet: Animal studies utilized male Nr4a1 knockout (NR4A1 −/− ) mice on a C57BL/6 background (Catalog S-KO-02468, Cyagen Biosciences) and 8-week-old male wild-type C57BL/6NCrl mice ((certificate No. HPPH-2022XS241, Beijing Vital River Laboratory Animal Technology Co., Ltd.).

Techniques: Expressing, Over Expression, Western Blot

Journal: iScience

Article Title: Estrogen upregulates NR4A1 to counter TGF beta induced pulmonary fibrosis therapeutic insights for IPF

doi: 10.1016/j.isci.2026.114756

Figure Lengend Snippet: TGF-β induces NR4A1 phosphorylation and alters its subcellular localization, thereby modulating fibrotic responses in lung fibroblasts (A) Immunoblotting analysis of p-NR4A1 and NR4A1 in MRC5 and HFL1 cells treated with increasing concentrations of TGF-β. Quantification shows a dose-dependent increase in p-NR4A1, with maximal induction at 20 ng/mL. (B) Time-course analysis of p-NR4A1 expression in MRC5 and HFL1 cells following stimulation with 20 ng/mL TGF-β. Phosphorylation increased progressively, became significant at 72 h, and peaked at 96 h. (C and D) Subcellular fractionation of MRC5 and HFL1 cells expressing wild-type NR4A1, NR4A1 ∧ S351A, or NR4A1 ∧ S351D under TGF-β stimulation. The S351D mutant was predominantly cytoplasmic, whereas the S351A mutant remained nuclear, indicating the phosphorylation-dependent redistribution of NR4A1. (E and F) Immunoblotting analysis of fibrotic markers, including α-SMA, SMAD2, fibronectin, and COL1A1 in MRC5 and HFL1 cells expressing wild-type NR4A1, NR4A1 ∧ S351A, or NR4A1 ∧ S351D. Quantification demonstrates that NR4A1 ∧ S351D enhances, whereas NR4A1 ∧ S351A attenuates, TGF-β-induced expression of fibrotic proteins. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; one-way ANOVA with post hoc tests was used for multiple group comparisons.

Article Snippet: Animal studies utilized male Nr4a1 knockout (NR4A1 −/− ) mice on a C57BL/6 background (Catalog S-KO-02468, Cyagen Biosciences) and 8-week-old male wild-type C57BL/6NCrl mice ((certificate No. HPPH-2022XS241, Beijing Vital River Laboratory Animal Technology Co., Ltd.).

Techniques: Phospho-proteomics, Western Blot, Expressing, Fractionation, Mutagenesis

Journal: iScience

Article Title: Estrogen upregulates NR4A1 to counter TGF beta induced pulmonary fibrosis therapeutic insights for IPF

doi: 10.1016/j.isci.2026.114756

Figure Lengend Snippet: TGF-β induced more severe lung fibrosis in NR4A1+ mice compared with control mice (A) Representative images of HE staining in mouse lung tissue (scale bars, 100 μm). Ashcroft scores were significantly increased in pNR4A1 + mice, while significantly decreased in pNR4A1 - mice. (B) Representative images of Masson staining in mouse lung tissue (scale bars, 100 μm), showing increased collagen deposition in pNR4A1 + mice and decreased collagen in pNR4A1 - mice. (C) Representative immunohistochemical images of α-SMA, SMAD2, fibronectin, and COL1A1 in mouse lung tissue (scale bars, 100 μm). (D) Quantification of immunohistochemical staining for α-SMA, SMAD2, fibronectin, and COL1A1. (E) Representative Western blot bands for α-SMA, SMAD2, fibronectin, and COL1A1 in mouse lung tissue. (F–I) Densitometric analysis of α-SMA, SMAD2, Fibronectin, and COL1A1 Western Blot bands in mouse lung tissue. Each group n = 6; ∗ p < 0.05, ∗∗ p < 0.01 compared to the two groups.

Article Snippet: Animal studies utilized male Nr4a1 knockout (NR4A1 −/− ) mice on a C57BL/6 background (Catalog S-KO-02468, Cyagen Biosciences) and 8-week-old male wild-type C57BL/6NCrl mice ((certificate No. HPPH-2022XS241, Beijing Vital River Laboratory Animal Technology Co., Ltd.).

Techniques: Control, Staining, Immunohistochemical staining, Western Blot

Journal: iScience

Article Title: Estrogen upregulates NR4A1 to counter TGF beta induced pulmonary fibrosis therapeutic insights for IPF

doi: 10.1016/j.isci.2026.114756

Figure Lengend Snippet: Estrogen promotes NR4A1 expression and inhibits fibrosis progression in IPF (A and B) Q-PCR and Western blot results show that NR4A1 expression increases gradually with prolonged E2 stimulation. (C and D) Q-PCR and immunoblotting results demonstrate that NR4A1 expression increases with increasing E2 concentrations. (E and F) Under TGF-β stimulation, mRNA levels of α-SMA, SMAD2, fibronectin, and COL1A1 were significantly increased, but E2 treatment significantly reduced the expression of these mRNAs. (G and H) immunoblotting results indicate that E2 alleviates TGF-β-induced fibrosis by decreasing the protein expression of α-SMA, SMAD2, fibronectin, and COL1A1. Each group n = 6; ∗ p < 0.05, ∗∗ p < 0.01 compared to the two groups.

Article Snippet: Animal studies utilized male Nr4a1 knockout (NR4A1 −/− ) mice on a C57BL/6 background (Catalog S-KO-02468, Cyagen Biosciences) and 8-week-old male wild-type C57BL/6NCrl mice ((certificate No. HPPH-2022XS241, Beijing Vital River Laboratory Animal Technology Co., Ltd.).

Techniques: Expressing, Western Blot

Journal: iScience

Article Title: Estrogen upregulates NR4A1 to counter TGF beta induced pulmonary fibrosis therapeutic insights for IPF

doi: 10.1016/j.isci.2026.114756

Figure Lengend Snippet: Silencing ERα signaling abolishes the E2-induced regulation of NR4A1 expression (A) q-PCR and immunoblotting results show that in MRC5 cells, silencing ERα prevents E2 from stimulating an increase in NR4A1 mRNA levels. (B) q-PCR and immunoblotting results show that in HFL1 cells, silencing ERα prevents E2 from stimulating an increase in NR4A1 protein expression. (C–F) Representative images and quantification of immunofluorescence for fibrosis-related factors α-SMA, SMAD2, fibronectin, and COL1A1 in cells ( n = 6, scale bars, 100 μm). (G–J) immunoblotting results indicate that in ERα siRNA-silenced cell lines, E2 cannot reduce protein levels of α-SMA, SMAD2, fibronectin, and COL1A1, and fails to inhibit TGF-β-induced fibrosis. Each group n = 6; ∗ p < 0.05, ∗∗ p < 0.01 compared to the two groups.

Article Snippet: Animal studies utilized male Nr4a1 knockout (NR4A1 −/− ) mice on a C57BL/6 background (Catalog S-KO-02468, Cyagen Biosciences) and 8-week-old male wild-type C57BL/6NCrl mice ((certificate No. HPPH-2022XS241, Beijing Vital River Laboratory Animal Technology Co., Ltd.).

Techniques: Expressing, Western Blot, Immunofluorescence

Journal: iScience

Article Title: Estrogen upregulates NR4A1 to counter TGF beta induced pulmonary fibrosis therapeutic insights for IPF

doi: 10.1016/j.isci.2026.114756

Figure Lengend Snippet: E2 alleviates pulmonary fibrosis in mice, while NR4A1 deficiency exacerbates the condition (A) Schematic illustration of estrogen capsule preparation. (B) Representative images of HE staining of mouse lung tissue ( n = 6 per group, scale bars, 100 μm) and Ashcroft fibrosis scores. (C) Representative images of Masson staining of mouse lung tissue ( n = 6 per group, scale bars, 100 μm) and collagen quantification analysis. (D) Representative images of immunohistochemical staining for α-SMA, SMAD2, Fibronectin, and COL1A1 in mouse lung tissue ( n = 6 per group, scale bars, 100 μm). (E–H) Quantification analysis of immunohistochemical staining for α-SMA, SMAD2, Fibronectin, and COL1A1 in mouse lung tissue. Each group n = 6; ∗ p < 0.05, ∗∗ p < 0.01 compared to the two groups.

Article Snippet: Animal studies utilized male Nr4a1 knockout (NR4A1 −/− ) mice on a C57BL/6 background (Catalog S-KO-02468, Cyagen Biosciences) and 8-week-old male wild-type C57BL/6NCrl mice ((certificate No. HPPH-2022XS241, Beijing Vital River Laboratory Animal Technology Co., Ltd.).

Techniques: Staining, Immunohistochemical staining

Journal: iScience

Article Title: Estrogen upregulates NR4A1 to counter TGF beta induced pulmonary fibrosis therapeutic insights for IPF

doi: 10.1016/j.isci.2026.114756

Figure Lengend Snippet: Subcutaneous placement of estrogen capsules in mice before TGF-β staining attenuates pulmonary fibrosis in mice (A) Schematic of the procedure for the subcutaneous implantation of estrogen capsules to treat IPF. (B) qPCR and immunoblotting analysis of NR4A1 expression in mice following estrogen capsule implantation. (C–N) Estrogen implantation reduces BLM-induced pulmonary fibrosis in mice. (C) Representative images of HE and Masson staining of mouse lung tissue ( n = 6 per group, scale bars, 100 μm). (D) Fibrosis scoring of lung tissue. (E) Collagen quantification analysis of lung tissue. (F–N) qPCR and immunoblotting analysis of α-SMA, SMAD2, Fibronectin (FN), and COL1A1 expression in lung tissue. Each group n = 6; ∗ p < 0.05, ∗∗ p < 0.01 compared to the two groups.

Article Snippet: Animal studies utilized male Nr4a1 knockout (NR4A1 −/− ) mice on a C57BL/6 background (Catalog S-KO-02468, Cyagen Biosciences) and 8-week-old male wild-type C57BL/6NCrl mice ((certificate No. HPPH-2022XS241, Beijing Vital River Laboratory Animal Technology Co., Ltd.).

Techniques: Capsules, Staining, Western Blot, Expressing

Journal: iScience

Article Title: Estrogen upregulates NR4A1 to counter TGF beta induced pulmonary fibrosis therapeutic insights for IPF

doi: 10.1016/j.isci.2026.114756

Figure Lengend Snippet: Comparing the therapeutic effects of estrogen capsule placement at the two time points, early placement of estrogen capsules was better (A) Representative Western blot bands of p-NR4A1 protein in mouse lung tissue under different interventions. (B) Quantification of p-NR4A1 protein bands by grayscale analysis. (C) Representative immunohistochemistry images of mouse lung tissue ( n = 6 per group, scale bars, 100 μm). (D–G) Immunohistochemical analysis of p-NR4A1 expression in mouse lung tissue, showing differences in expression levels across different interventions. Each group n = 6; ∗ p < 0.05, ∗∗ p < 0.01 compared to the two groups.

Article Snippet: Animal studies utilized male Nr4a1 knockout (NR4A1 −/− ) mice on a C57BL/6 background (Catalog S-KO-02468, Cyagen Biosciences) and 8-week-old male wild-type C57BL/6NCrl mice ((certificate No. HPPH-2022XS241, Beijing Vital River Laboratory Animal Technology Co., Ltd.).

Techniques: Capsules, Western Blot, Immunohistochemistry, Immunohistochemical staining, Expressing

Journal: iScience

Article Title: Estrogen upregulates NR4A1 to counter TGF beta induced pulmonary fibrosis therapeutic insights for IPF

doi: 10.1016/j.isci.2026.114756

Figure Lengend Snippet: Regulatory role of NR4A1 in TGF-β signaling in normal, IPF, and estrogen treatment conditions (A) Under normal physiological conditions, brief TGF-β stimulation induces the upregulation of NR4A1. NR4A1 then suppresses epithelial-mesenchymal transition (EMT) and collagen deposition via a negative feedback mechanism. (B) In the IPF state, sustained TGF-β signaling leads to prolonged EMT and collagen deposition, while promoting NR4A1 phosphorylation, which disables its negative feedback regulation of TGF-β signaling. (C) Estrogen treatment enhances NR4A1 transcription and inhibits NR4A1 phosphorylation, thereby restoring its negative feedback regulation on TGF-β signaling.

Article Snippet: Animal studies utilized male Nr4a1 knockout (NR4A1 −/− ) mice on a C57BL/6 background (Catalog S-KO-02468, Cyagen Biosciences) and 8-week-old male wild-type C57BL/6NCrl mice ((certificate No. HPPH-2022XS241, Beijing Vital River Laboratory Animal Technology Co., Ltd.).

Techniques: Phospho-proteomics

Journal: iScience

Article Title: Estrogen upregulates NR4A1 to counter TGF beta induced pulmonary fibrosis therapeutic insights for IPF

doi: 10.1016/j.isci.2026.114756

Figure Lengend Snippet: Abnormal expression of NR4A1, p-NR4A1, and ER in lung tissues of patients with IPF (A–C) qPCR analysis of NR4A1, ERα, and ERβ mRNA expression in lung tissues from healthy controls (Ctrl, n = 11) and patients with IPF ( n = 23). (D) Representative immunoblotting images showing protein levels of NR4A1, p-NR4A1, ERα, and ERβ in lung tissues from Ctrl and IPF groups. β-Actin served as a loading control. (E) Quantification of immunoblotting results for NR4A1, p-NR4A1, p-NR4A1/NR4A1 ratio, ERα, and ERβ. (F–I) Correlation analysis between NR4A1 and ERα (F), NR4A1 and ERβ (G), p-NR4A1 and ERα (H), and p-NR4A1 and ERβ (I) in IPF lung tissues. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001; Student’s t test for group comparisons, Pearson’s correlation for association analysis.

Article Snippet: Nr4a1 knockout (NR4A1−/−) mice on a C57BL/6 background (KOCMP-15370-Nr4a1-B6N-VA) , Cyagen Biosciences , Cat# S-KO-02468.

Techniques: Expressing, Western Blot, Control

Journal: iScience

Article Title: Estrogen upregulates NR4A1 to counter TGF beta induced pulmonary fibrosis therapeutic insights for IPF

doi: 10.1016/j.isci.2026.114756

Figure Lengend Snippet: Silencing of NR4A1 in cells exacerbates TGF-β-induced fibrosis and increases the expression of α-SMA, SMAD2, fibronectin, and COL1A1 (A and B) qPCR and Western Blot analyses confirm reduced mRNA and protein expression levels of NR4A1 in MRC5 and HFL1 cells following siRNA-mediated NR4A1 silencing. (C and D) qPCR analysis of α-SMA, SMAD2, Fibronectin, and COL1A1 expression in NR4A1-silenced cells after TGF-β stimulation, showing elevated levels indicative of enhanced fibrosis. (E and F) Western blot analysis further confirms increased expression of α-SMA, SMAD2, fibronectin, and COL1A1 in NR4A1-silenced cells upon TGF-β stimulation, demonstrating that NR4A1 downregulation aggravates fibrotic responses. Each group n = 6; ∗ p < 0.05, ∗∗ p < 0.01 compared to the two groups.

Article Snippet: Nr4a1 knockout (NR4A1−/−) mice on a C57BL/6 background (KOCMP-15370-Nr4a1-B6N-VA) , Cyagen Biosciences , Cat# S-KO-02468.

Techniques: Expressing, Western Blot

Journal: iScience

Article Title: Estrogen upregulates NR4A1 to counter TGF beta induced pulmonary fibrosis therapeutic insights for IPF

doi: 10.1016/j.isci.2026.114756

Figure Lengend Snippet: Increasing NR4A1 expression in cells attenuates TGF-β-induced fibrosis and decreases α-SMA, SMAD2, Fibronectin, and COL1A1 expression (A and B) NR4A1 overexpression in MRC5 and HFL1 cells induced by tetracycline, confirmed by increased NR4A1 mRNA and protein levels through qPCR and Western blot analyses. (C–J) qPCR analysis shows reduced expression levels of fibrosis markers α-SMA, SMAD2, Fibronectin, and COL1A1 in NR4A1-overexpressing cells following TGF-β stimulation, indicating diminished fibrotic responses. (K and L) Western blot analysis further confirms decreased protein levels of α-SMA, SMAD2, Fibronectin, and COL1A1 in TGF-β-stimulated NR4A1-overexpressing cells, demonstrating the antifibrotic effect of NR4A1 upregulation. Each group n = 6; ∗ p < 0.05, ∗∗ p < 0.01 compared to the two groups.

Article Snippet: Nr4a1 knockout (NR4A1−/−) mice on a C57BL/6 background (KOCMP-15370-Nr4a1-B6N-VA) , Cyagen Biosciences , Cat# S-KO-02468.

Techniques: Expressing, Over Expression, Western Blot

Journal: iScience

Article Title: Estrogen upregulates NR4A1 to counter TGF beta induced pulmonary fibrosis therapeutic insights for IPF

doi: 10.1016/j.isci.2026.114756

Figure Lengend Snippet: TGF-β induces NR4A1 phosphorylation and alters its subcellular localization, thereby modulating fibrotic responses in lung fibroblasts (A) Immunoblotting analysis of p-NR4A1 and NR4A1 in MRC5 and HFL1 cells treated with increasing concentrations of TGF-β. Quantification shows a dose-dependent increase in p-NR4A1, with maximal induction at 20 ng/mL. (B) Time-course analysis of p-NR4A1 expression in MRC5 and HFL1 cells following stimulation with 20 ng/mL TGF-β. Phosphorylation increased progressively, became significant at 72 h, and peaked at 96 h. (C and D) Subcellular fractionation of MRC5 and HFL1 cells expressing wild-type NR4A1, NR4A1 ∧ S351A, or NR4A1 ∧ S351D under TGF-β stimulation. The S351D mutant was predominantly cytoplasmic, whereas the S351A mutant remained nuclear, indicating the phosphorylation-dependent redistribution of NR4A1. (E and F) Immunoblotting analysis of fibrotic markers, including α-SMA, SMAD2, fibronectin, and COL1A1 in MRC5 and HFL1 cells expressing wild-type NR4A1, NR4A1 ∧ S351A, or NR4A1 ∧ S351D. Quantification demonstrates that NR4A1 ∧ S351D enhances, whereas NR4A1 ∧ S351A attenuates, TGF-β-induced expression of fibrotic proteins. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; one-way ANOVA with post hoc tests was used for multiple group comparisons.

Article Snippet: Nr4a1 knockout (NR4A1−/−) mice on a C57BL/6 background (KOCMP-15370-Nr4a1-B6N-VA) , Cyagen Biosciences , Cat# S-KO-02468.

Techniques: Phospho-proteomics, Western Blot, Expressing, Fractionation, Mutagenesis

Journal: iScience

Article Title: Estrogen upregulates NR4A1 to counter TGF beta induced pulmonary fibrosis therapeutic insights for IPF

doi: 10.1016/j.isci.2026.114756

Figure Lengend Snippet: TGF-β induced more severe lung fibrosis in NR4A1+ mice compared with control mice (A) Representative images of HE staining in mouse lung tissue (scale bars, 100 μm). Ashcroft scores were significantly increased in pNR4A1 + mice, while significantly decreased in pNR4A1 - mice. (B) Representative images of Masson staining in mouse lung tissue (scale bars, 100 μm), showing increased collagen deposition in pNR4A1 + mice and decreased collagen in pNR4A1 - mice. (C) Representative immunohistochemical images of α-SMA, SMAD2, fibronectin, and COL1A1 in mouse lung tissue (scale bars, 100 μm). (D) Quantification of immunohistochemical staining for α-SMA, SMAD2, fibronectin, and COL1A1. (E) Representative Western blot bands for α-SMA, SMAD2, fibronectin, and COL1A1 in mouse lung tissue. (F–I) Densitometric analysis of α-SMA, SMAD2, Fibronectin, and COL1A1 Western Blot bands in mouse lung tissue. Each group n = 6; ∗ p < 0.05, ∗∗ p < 0.01 compared to the two groups.

Article Snippet: Nr4a1 knockout (NR4A1−/−) mice on a C57BL/6 background (KOCMP-15370-Nr4a1-B6N-VA) , Cyagen Biosciences , Cat# S-KO-02468.

Techniques: Control, Staining, Immunohistochemical staining, Western Blot

Journal: iScience

Article Title: Estrogen upregulates NR4A1 to counter TGF beta induced pulmonary fibrosis therapeutic insights for IPF

doi: 10.1016/j.isci.2026.114756

Figure Lengend Snippet: Estrogen promotes NR4A1 expression and inhibits fibrosis progression in IPF (A and B) Q-PCR and Western blot results show that NR4A1 expression increases gradually with prolonged E2 stimulation. (C and D) Q-PCR and immunoblotting results demonstrate that NR4A1 expression increases with increasing E2 concentrations. (E and F) Under TGF-β stimulation, mRNA levels of α-SMA, SMAD2, fibronectin, and COL1A1 were significantly increased, but E2 treatment significantly reduced the expression of these mRNAs. (G and H) immunoblotting results indicate that E2 alleviates TGF-β-induced fibrosis by decreasing the protein expression of α-SMA, SMAD2, fibronectin, and COL1A1. Each group n = 6; ∗ p < 0.05, ∗∗ p < 0.01 compared to the two groups.

Article Snippet: Nr4a1 knockout (NR4A1−/−) mice on a C57BL/6 background (KOCMP-15370-Nr4a1-B6N-VA) , Cyagen Biosciences , Cat# S-KO-02468.

Techniques: Expressing, Western Blot

Journal: iScience

Article Title: Estrogen upregulates NR4A1 to counter TGF beta induced pulmonary fibrosis therapeutic insights for IPF

doi: 10.1016/j.isci.2026.114756

Figure Lengend Snippet: Silencing ERα signaling abolishes the E2-induced regulation of NR4A1 expression (A) q-PCR and immunoblotting results show that in MRC5 cells, silencing ERα prevents E2 from stimulating an increase in NR4A1 mRNA levels. (B) q-PCR and immunoblotting results show that in HFL1 cells, silencing ERα prevents E2 from stimulating an increase in NR4A1 protein expression. (C–F) Representative images and quantification of immunofluorescence for fibrosis-related factors α-SMA, SMAD2, fibronectin, and COL1A1 in cells ( n = 6, scale bars, 100 μm). (G–J) immunoblotting results indicate that in ERα siRNA-silenced cell lines, E2 cannot reduce protein levels of α-SMA, SMAD2, fibronectin, and COL1A1, and fails to inhibit TGF-β-induced fibrosis. Each group n = 6; ∗ p < 0.05, ∗∗ p < 0.01 compared to the two groups.

Article Snippet: Nr4a1 knockout (NR4A1−/−) mice on a C57BL/6 background (KOCMP-15370-Nr4a1-B6N-VA) , Cyagen Biosciences , Cat# S-KO-02468.

Techniques: Expressing, Western Blot, Immunofluorescence

Journal: iScience

Article Title: Estrogen upregulates NR4A1 to counter TGF beta induced pulmonary fibrosis therapeutic insights for IPF

doi: 10.1016/j.isci.2026.114756

Figure Lengend Snippet: E2 alleviates pulmonary fibrosis in mice, while NR4A1 deficiency exacerbates the condition (A) Schematic illustration of estrogen capsule preparation. (B) Representative images of HE staining of mouse lung tissue ( n = 6 per group, scale bars, 100 μm) and Ashcroft fibrosis scores. (C) Representative images of Masson staining of mouse lung tissue ( n = 6 per group, scale bars, 100 μm) and collagen quantification analysis. (D) Representative images of immunohistochemical staining for α-SMA, SMAD2, Fibronectin, and COL1A1 in mouse lung tissue ( n = 6 per group, scale bars, 100 μm). (E–H) Quantification analysis of immunohistochemical staining for α-SMA, SMAD2, Fibronectin, and COL1A1 in mouse lung tissue. Each group n = 6; ∗ p < 0.05, ∗∗ p < 0.01 compared to the two groups.

Article Snippet: Nr4a1 knockout (NR4A1−/−) mice on a C57BL/6 background (KOCMP-15370-Nr4a1-B6N-VA) , Cyagen Biosciences , Cat# S-KO-02468.

Techniques: Staining, Immunohistochemical staining

Journal: iScience

Article Title: Estrogen upregulates NR4A1 to counter TGF beta induced pulmonary fibrosis therapeutic insights for IPF

doi: 10.1016/j.isci.2026.114756

Figure Lengend Snippet: Subcutaneous placement of estrogen capsules in mice before TGF-β staining attenuates pulmonary fibrosis in mice (A) Schematic of the procedure for the subcutaneous implantation of estrogen capsules to treat IPF. (B) qPCR and immunoblotting analysis of NR4A1 expression in mice following estrogen capsule implantation. (C–N) Estrogen implantation reduces BLM-induced pulmonary fibrosis in mice. (C) Representative images of HE and Masson staining of mouse lung tissue ( n = 6 per group, scale bars, 100 μm). (D) Fibrosis scoring of lung tissue. (E) Collagen quantification analysis of lung tissue. (F–N) qPCR and immunoblotting analysis of α-SMA, SMAD2, Fibronectin (FN), and COL1A1 expression in lung tissue. Each group n = 6; ∗ p < 0.05, ∗∗ p < 0.01 compared to the two groups.

Article Snippet: Nr4a1 knockout (NR4A1−/−) mice on a C57BL/6 background (KOCMP-15370-Nr4a1-B6N-VA) , Cyagen Biosciences , Cat# S-KO-02468.

Techniques: Capsules, Staining, Western Blot, Expressing

Journal: iScience

Article Title: Estrogen upregulates NR4A1 to counter TGF beta induced pulmonary fibrosis therapeutic insights for IPF

doi: 10.1016/j.isci.2026.114756

Figure Lengend Snippet: Comparing the therapeutic effects of estrogen capsule placement at the two time points, early placement of estrogen capsules was better (A) Representative Western blot bands of p-NR4A1 protein in mouse lung tissue under different interventions. (B) Quantification of p-NR4A1 protein bands by grayscale analysis. (C) Representative immunohistochemistry images of mouse lung tissue ( n = 6 per group, scale bars, 100 μm). (D–G) Immunohistochemical analysis of p-NR4A1 expression in mouse lung tissue, showing differences in expression levels across different interventions. Each group n = 6; ∗ p < 0.05, ∗∗ p < 0.01 compared to the two groups.

Article Snippet: Nr4a1 knockout (NR4A1−/−) mice on a C57BL/6 background (KOCMP-15370-Nr4a1-B6N-VA) , Cyagen Biosciences , Cat# S-KO-02468.

Techniques: Capsules, Western Blot, Immunohistochemistry, Immunohistochemical staining, Expressing

Journal: iScience

Article Title: Estrogen upregulates NR4A1 to counter TGF beta induced pulmonary fibrosis therapeutic insights for IPF

doi: 10.1016/j.isci.2026.114756

Figure Lengend Snippet: Regulatory role of NR4A1 in TGF-β signaling in normal, IPF, and estrogen treatment conditions (A) Under normal physiological conditions, brief TGF-β stimulation induces the upregulation of NR4A1. NR4A1 then suppresses epithelial-mesenchymal transition (EMT) and collagen deposition via a negative feedback mechanism. (B) In the IPF state, sustained TGF-β signaling leads to prolonged EMT and collagen deposition, while promoting NR4A1 phosphorylation, which disables its negative feedback regulation of TGF-β signaling. (C) Estrogen treatment enhances NR4A1 transcription and inhibits NR4A1 phosphorylation, thereby restoring its negative feedback regulation on TGF-β signaling.

Article Snippet: Nr4a1 knockout (NR4A1−/−) mice on a C57BL/6 background (KOCMP-15370-Nr4a1-B6N-VA) , Cyagen Biosciences , Cat# S-KO-02468.

Techniques: Phospho-proteomics