Journal: International Journal of Molecular Sciences

Article Title: NR4A1 Mediates Bronchopulmonary Dysplasia-Like Lung Injury Induced by Intrauterine Inflammation in Mouse Offspring

doi: 10.3390/ijms26146931

Figure Lengend Snippet: Increased NR4A1 expression was associated with impaired lung development induced by IUI. ( a ) Representative image of hematoxylin- and eosin-stained lung tissues from C57BL/6 neonatal mice exposed to IUI and control mice on postnatal day 1. Scale bars, 100 μm ( n = 6–7). ( b ) Quantification of alveolar size for ( a ). ( c ) Quantification of alveolar number for ( a ). ( d ) Western Blot analysis of NR4A1 expression in lung tissues from C57BL/6 neonatal mice exposed to IUI and control mice on postnatal day 1. ( e ) The relative protein level of NR4A1 was quantified for ( d ), with GAPDH used as the loading control ( n = 7). ( f ) Immunohistochemistry analysis of NR4A1 expression in lung tissues from C57BL/6 neonatal mice exposed to IUI and control mice on postnatal day 1. Scale bars, 100 μm ( n = 6–7). ( g ) Quantification of NR4A1-positive areas for ( f ). ( h ) Representative image of hematoxylin and eosin-stained lung tissues from C57BL/6 neonatal mice exposed to IUI and control mice at 3 months postnatal. Scale bars, 100 μm ( n = 11–12). ( i ) Quantification of alveolar size for ( h ). ( j ) Quantification of alveolar number for ( h ). All data are presented as the mean ± SD. Statistical analysis was performed using unpaired Student’s t tests. * p < 0.05, ** p < 0.01 as indicated. Arrows in subfigure (f) indicate NR4A1-positive cells.

Article Snippet: The sections were probed with rabbit anti-NR4A1 antibodies (1:350; Novus, St. Louis, MI, USA) in the incubator overnight at 4 °C.

Techniques: Expressing, Staining, Control, Western Blot, Immunohistochemistry

Journal: International Journal of Molecular Sciences

Article Title: NR4A1 Mediates Bronchopulmonary Dysplasia-Like Lung Injury Induced by Intrauterine Inflammation in Mouse Offspring

doi: 10.3390/ijms26146931

Figure Lengend Snippet: Involvement of NR4A1 in Lung Injury Induced by IUI. The efficiency of NR4A1 RNA interference was evaluated by RT-qPCR ( a ) and Western blot ( b ). Representative image of lung tissues from C57BL/6 neonatal mice exposed to IUI on postnatal day 7, with NR4A1 siRNA administered intranasally from postnatal day 2 to 5, stained with hematoxylin and eosin. Scale bars, 200 μm ( n = 11–20) ( c ). ( d ) Quantification of alveolar size for ( c ). ( e ) Quantification of alveoli number for ( c ). All data are presented as the mean ± SD. Statistical analysis was performed using unpaired Student’s t tests. * p < 0.05, ** p < 0.01 as indicated.

Article Snippet: The sections were probed with rabbit anti-NR4A1 antibodies (1:350; Novus, St. Louis, MI, USA) in the incubator overnight at 4 °C.

Techniques: Quantitative RT-PCR, Western Blot, Staining

Journal: International Journal of Molecular Sciences

Article Title: NR4A1 Mediates Bronchopulmonary Dysplasia-Like Lung Injury Induced by Intrauterine Inflammation in Mouse Offspring

doi: 10.3390/ijms26146931

Figure Lengend Snippet: Differential transcriptome and proliferation caused by overexpression of NR4A1 in MLE-12 cells. The efficiency of NR4A1 overexpression was evaluated by Western blot ( a ). ( b ) Representative images of EdU-positive cells in NR4A1-overexpressing and control MLE-12 cells ( n = 6). ( c ) The quantification of EdU-positive cells for ( b ). ( d ) A volcano plot of DEGs in MLE-12 cells overexpressing NR4A1. ( e ) The top 15 DEGs associated with lung injury induced by NR4A1 overexpression. ( f , g ) The DEGs significantly regulated by NR4A1 overexpression are enriched in the top 30 KEGG and GO pathways. ( h ) Western Blot analysis of whole-cell lysates prepared from NR4A1-overexpressing and control MLE-12 cells after transfection with the vectors for 48 h. The relative protein level of pERK1/2/tERK1/2 ( i ) and pAKT/tAKT ( j ) were quantified for ( h ), with GAPDH used as the loading control (n = 9). All data are presented as the mean ± SD. Statistical analysis was performed using unpaired Student’s t test. * p < 0.05, ** p < 0.01, and *** p < 0.001 as indicated. DAPI: 4′,6-diamidino-2-phenylindole; EdU: 5-ethynyl-2-deoxyuridine; DEGs: differentially expressed genes.

Article Snippet: The sections were probed with rabbit anti-NR4A1 antibodies (1:350; Novus, St. Louis, MI, USA) in the incubator overnight at 4 °C.

Techniques: Over Expression, Western Blot, Control, Transfection

Journal: International Journal of Molecular Sciences

Article Title: NR4A1 Mediates Bronchopulmonary Dysplasia-Like Lung Injury Induced by Intrauterine Inflammation in Mouse Offspring

doi: 10.3390/ijms26146931

Figure Lengend Snippet: EREG is a key downstream target of NR4A1. ( a ) Verification of DEGs associated with lung injury in MLE-12 cells after transfection with the vectors for 48 h by RT-qPCR (n = 6–8). ( b ) Western Blot analysis of whole-cell lysates prepared from NR4A1-overexpressing and control MLE-12 cells after transfection with the vectors for 48 h. ( c ) The relative protein level of EREG in ( b ) (n = 5). ( d ) ELISA analysis of the protein levels of EREG in the lung tissues of neonatal mice with IUI on postnatal day 1 (n = 11). ( e ) Schematic illustration of predicted NR4A1 binding elements (S1–S3) in the total 2000 bp sequence of the promoter and 5′UTR of the murine Ereg gene. ( f ) Effect of S1–3 fragments EREG transcription activity mediated by NR4A1 (n = 6). All data are presented as the mean ± SD. The data were analyzed using unpaired t -tests. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, # p < 0.05; ## p < 0.01, and #### p < 0.0001 as indicated.

Article Snippet: The sections were probed with rabbit anti-NR4A1 antibodies (1:350; Novus, St. Louis, MI, USA) in the incubator overnight at 4 °C.

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Binding Assay, Sequencing, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: NR4A1 Mediates Bronchopulmonary Dysplasia-Like Lung Injury Induced by Intrauterine Inflammation in Mouse Offspring

doi: 10.3390/ijms26146931

Figure Lengend Snippet: NR4A1-EREG-EGFR signaling pathway involved in development of pulmonary fibrosis induced by IUI. ( a ) Representative image of collagen fiber staining using Masson’s trichrome staining in the lungs of neonatal mice with IUI on postnatal day 1 ( n = 7–9). ( b ) Quantification of Masson-positive areas for ( b ). ( c ) Representative images of collagen fiber staining using Masson’s trichrome staining in the lungs of neonatal mice with IUI at 6 months postnatal ( n = 4–5). ( d ) Quantification of Masson-positive areas for ( c ). EMT-related gene expression was analyzed by RT-qPCR in MLE-12 cells ( e ) and A549 cells ( j ) after 72h transfection with NR4A1-overexpressing vectors (n = 4–5). EMT-related gene expression was analyzed in L929 cells ( f ) by RT-qPCR following 24 h treatment with conditioned media derived from NR4A1-overexpressing MLE-12 cells cultured for 48 h ( n = 3–4). EMT-related gene expression was analyzed using RT-qPCR in MLE-12 cells ( g ) and A549 cells ( k ) pretreated with gefitinib (10 μmoL) for 1 h, followed by EREG (50 ng/mL) for 12 h ( n = 3–5). ( h ) Western Blot analysis of whole-cell lysates prepared from NR4A1-overexpressing and control MLE-12 cells treated with DMSO or gefitinib (10 μmoL), respectively, after transfection with the vectors for 48 h. ( i ) The relative protein levels of pERK1/2/tERK1/2 and pAKT/tAKT were quantified for ( h ), with GAPDH used as the loading control ( n = 3). All data are presented as the mean ± SD. The data were analyzed using unpaired t -tests. * p < 0.05, ** p <0.01, *** p <0.001 as indicated.

Article Snippet: The sections were probed with rabbit anti-NR4A1 antibodies (1:350; Novus, St. Louis, MI, USA) in the incubator overnight at 4 °C.

Techniques: Staining, Gene Expression, Quantitative RT-PCR, Transfection, Derivative Assay, Cell Culture, Western Blot, Control