cleaved parp1  (Cell Signaling Technology Inc)

Journal: Science Advances

Article Title: PARP1 stabilizes FOXN3 to suppress pulmonary fibrosis through p38-related feedback regulation

doi: 10.1126/sciadv.ady1681

Figure Lengend Snippet: ( A ) Affinity-purified immunoprecipitates from HEK293T cells via anti-Flag immunoprecipitation (IP) were analyzed by mass spectrometry to identify unique peptides associated with Flag-tagged FOXN3. The sequences of the PARP1 peptides linked to FOXN3 are presented. ( B ) A co-IP assay was carried out in A549 cells to examine the endogenous association between FOXN3 and PARP1. ( C ) An anti-Flag co-IP assay was performed in A549 cells stably expressing Flag-tagged FOXN3 to assess the association between FOXN3 and PARP1 upon TGF-β treatment (10 ng/ml) at the indicated time points [0, 16, 24, and 48 hours (hr)]. IB, immunoblot. The cells were treated with MG-132 (20 μM, 4 hours) before collection. ( D ) An immunofluorescence assay was performed in A549 cells to assess the colocalization of FOXN3 and PARP1. Scale bar, 10 μm. ( E ) A deletion-mapping assay was performed in HEK293T cells to define the region within FOXN3 associated with PARP1. Flag-tagged FOXN3 WT or its various mutants were cotransfected with HA-tagged PARP1 into HEK293T cells (ΔNTD: Δ1-113aa; ΔForkhead: Δ114-199aa; ΔCTD: Δ200-490aa). ( F ) An in vitro GST pull-down assay was performed to examine the direct interaction between bacterially expressed GST-tagged FOXN3 and His-tagged PARP1 purified from Sf9 insect cells. ( G ) Venn diagrams illustrating the overlapping genes cotargeted by FOXN3 and PARP1 in A549 cells, as determined via CUT&Tag data analysis. ( H ) The normalized distribution of reads for the FOXN3 and PARP1 peaks at the transcription start site (TSS) is presented. ( I ) The binding profiles of FOXN3 and PARP1 at representative Smad response genes in A549 cells were analyzed via CUT&Tag data. The blotting data for (B), (C), (E), and (F) were quantified as the means from three independent experiments using ImageJ software. The data for (C) and (E) were analyzed using one-way ANOVA. ** P < 0.01 and *** P < 0.001.

Article Snippet: The primary antibodies used for Western blotting were as follows: anti–hemagglutinin (HA) (1:2000, no. 51064-2-AP, Proteintech), anti-Smad4 (1:1000, no. 10231-1-AP, Proteintech), anti-FOXN3 (1:1000, no. 25399-1-AP, Proteintech), anti-Flag (1:1000, no. 20543-1-AP, Proteintech), anti–GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (1:2000, no. 2118, Cell Signaling Technology), anti–α-Tubulin (1:2000, no. 11224-1-AP, Proteintech), anti-FOXN3 (1:1000, no. ab129453, Abcam), anti-PARP1 (1:1000, no. 66520-1-Ig, Proteintech), anti-p38 (1:1000, no. 14064-1-AP, Proteintech), anti–α-SMA (1:500, no. 14395-1-AP, Proteintech), anti-HSP27 (1:500, no. 18284-1-AP, Proteintech), anti–Phospho-HSP27-S78 (1:500, no. 28900-1-AP, Proteintech), anti-FOXN3 (1:500, no. A15039, ABclonal), anti–Cleaved Caspase3 (1:500, no. 9661, Cell Signaling Technology), anti–Cleaved PARP1 (1:500, no. 5625, Cell Signaling Technology), and anti–phospho-FOXN3 S83/85 (1:1000).

Techniques: Affinity Purification, Immunoprecipitation, Mass Spectrometry, Co-Immunoprecipitation Assay, Stable Transfection, Expressing, Western Blot, Immunofluorescence, Mapping Assay, In Vitro, Pull Down Assay, Purification, Binding Assay, Software

Journal: Science Advances

Article Title: PARP1 stabilizes FOXN3 to suppress pulmonary fibrosis through p38-related feedback regulation

doi: 10.1126/sciadv.ady1681

Figure Lengend Snippet: ( A ) Western blot analysis was conducted to evaluate the levels of PARP1 and FOXN3 in the total lysate of A549 cells treated with TGF-β (10 ng/ml) at the specified time points (0, 16, 24, and 48 hours). TCL, total cell lysate. ( B ) Western blotting measured PARP1 and FOXN3 in the lungs of mice with or without BLM treatment (1.5 mg/kg, 21 days) ( n = 3). ( C ) Western blotting assessed PARP1 and FOXN3 levels in WT and Parp1 −/− A549 cells. ( D ) Western blotting evaluated PARP1 and FOXN3 in the lungs of WT or Parp1 fl/fl mice treated with or without adenovirus-expressed Cre recombinase ( n = 4). ( E ) Western blot analysis was conducted to measure the levels of FOXN3 in Parp1 −/− A549 cells reintroduced with exogenous WT PARP1 or its E988A inactive mutant form. ( F ) Western blot analysis was conducted to measure the levels of FOXN3 in A549 cells treated with BYK204165 (20 μM) or PJ34 (5 μM) at the indicated time points. ( G ) CUT&Tag analyses showing the binding profiles of FOXN3 to Smad response genes in A549 cells with and without BYK204165 treatment (20 μM, 4 hours). ( H ) ChIP assays were performed to detect the distribution of FOXN3 at representative Smad response genes in A549 cells, both with and without BYK204165 treatment (20 μM, 4 hours). ( I ) ChIP assays evaluated Smad protein distribution in WT and Parp1 −/− A549 cells treated with TGF-β (10 ng/ml) for 16 hours. ( J ) Quantitative PCR analyzed Smad response gene expression in WT and Parp1 −/− A549 cells treated with TGF-β (10 ng/ml) for 16 hours. The data in (H) to (J) were assessed by a two-tailed Student’s t test and are shown as the means ± SD. The blotting data for (A), (E), and (F) were quantified as the means from three independent experiments using ImageJ software. Data for (A), (E), and (F) were analyzed using one-way ANOVA, while data for (B) to (D) were assessed using two-tailed Student’s t tests. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: The primary antibodies used for Western blotting were as follows: anti–hemagglutinin (HA) (1:2000, no. 51064-2-AP, Proteintech), anti-Smad4 (1:1000, no. 10231-1-AP, Proteintech), anti-FOXN3 (1:1000, no. 25399-1-AP, Proteintech), anti-Flag (1:1000, no. 20543-1-AP, Proteintech), anti–GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (1:2000, no. 2118, Cell Signaling Technology), anti–α-Tubulin (1:2000, no. 11224-1-AP, Proteintech), anti-FOXN3 (1:1000, no. ab129453, Abcam), anti-PARP1 (1:1000, no. 66520-1-Ig, Proteintech), anti-p38 (1:1000, no. 14064-1-AP, Proteintech), anti–α-SMA (1:500, no. 14395-1-AP, Proteintech), anti-HSP27 (1:500, no. 18284-1-AP, Proteintech), anti–Phospho-HSP27-S78 (1:500, no. 28900-1-AP, Proteintech), anti-FOXN3 (1:500, no. A15039, ABclonal), anti–Cleaved Caspase3 (1:500, no. 9661, Cell Signaling Technology), anti–Cleaved PARP1 (1:500, no. 5625, Cell Signaling Technology), and anti–phospho-FOXN3 S83/85 (1:1000).

Techniques: Western Blot, Mutagenesis, Binding Assay, Real-time Polymerase Chain Reaction, Gene Expression, Two Tailed Test, Software

Journal: Science Advances

Article Title: PARP1 stabilizes FOXN3 to suppress pulmonary fibrosis through p38-related feedback regulation

doi: 10.1126/sciadv.ady1681

Figure Lengend Snippet: ( A ) Western blot analysis showing PARP1 protein levels in the lungs of Parp1 fl/fl mice with and without Cre recombinase treatment ( n = 3). ( B ) Volcano plot of RNA sequencing data depicting the differential expression of genes in the lungs of mice following conditional KO of PARP1. ( C ) Heatmap analyses of RNA sequencing data showing the changes in the expression of Smad downstream-regulated genes in the lungs of mice following conditional KO of PARP1. ( D ) Histological trichrome staining comparing the fibrotic response in the lungs of Parp1 fl/fl mice with and without Cre recombinase treatment following BLM (1.5 mg/kg, 21 days) or vehicle control (PBS) treatment. Scale bar, 100 μm. The collagen content from four distinct fields for each sample was quantified using ImageJ software and expressed as a percentage of the total area. ( E ) H&E staining analysis was performed to evaluate the inflammatory response in the lungs of Parp1 fl/fl mice with and without Cre recombinase treatment following BLM (1.5 mg/kg, 21 days) or vehicle control (PBS) treatment. Scale bar, 100 μm. ( F ) ELISAs were performed to measure TNFα and IL-6 levels in BAL fluid from Parp1 fl/fl mice with and without Cre recombinase treatment following BLM or vehicle control (PBS) treatment ( n = 4). ( G ) Quantitative PCR analysis was performed to assess the RNA levels of profibrotic factors in the lungs of Parp1 fl/fl mice with and without Cre recombinase treatment following BLM (1.5 mg/kg, 21 days) or vehicle control (PBS) treatment ( n = 4). ( H ) Hydroxyproline measurements comparing the collagen content in the lungs of Parp1 fl/fl mice with and without Cre recombinase treatment in the presence or absence of BLM stimulation (1.5 mg/kg, 21 days) ( n = 4). ( I ) Anti-ProSPC and anti–α-SMA immunofluorescence staining of lung sections from Parp1 fl/fl mice with and without Cre recombinase treatment following BLM treatment (1.5 mg/kg, 21 days) ( n = 4) was analyzed. Fluorescence intensity from four fields per sample was quantified using ImageJ and expressed as relative intensity. DAPI, 4′,6-diamidino-2-phenylindole. The data for (F) to (H) were assessed using one-way ANOVA, while data for (I) were analyzed using two-tailed Student’s t tests, and all results are presented as the means ± SDs. The blotting data for (A) were quantified as the means from three independent experiments using ImageJ software and analyzed using two-tailed Student’s t tests. * P < 0.05.

Article Snippet: The primary antibodies used for Western blotting were as follows: anti–hemagglutinin (HA) (1:2000, no. 51064-2-AP, Proteintech), anti-Smad4 (1:1000, no. 10231-1-AP, Proteintech), anti-FOXN3 (1:1000, no. 25399-1-AP, Proteintech), anti-Flag (1:1000, no. 20543-1-AP, Proteintech), anti–GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (1:2000, no. 2118, Cell Signaling Technology), anti–α-Tubulin (1:2000, no. 11224-1-AP, Proteintech), anti-FOXN3 (1:1000, no. ab129453, Abcam), anti-PARP1 (1:1000, no. 66520-1-Ig, Proteintech), anti-p38 (1:1000, no. 14064-1-AP, Proteintech), anti–α-SMA (1:500, no. 14395-1-AP, Proteintech), anti-HSP27 (1:500, no. 18284-1-AP, Proteintech), anti–Phospho-HSP27-S78 (1:500, no. 28900-1-AP, Proteintech), anti-FOXN3 (1:500, no. A15039, ABclonal), anti–Cleaved Caspase3 (1:500, no. 9661, Cell Signaling Technology), anti–Cleaved PARP1 (1:500, no. 5625, Cell Signaling Technology), and anti–phospho-FOXN3 S83/85 (1:1000).

Techniques: Western Blot, RNA Sequencing, Quantitative Proteomics, Expressing, Staining, Control, Software, Real-time Polymerase Chain Reaction, Immunofluorescence, Fluorescence, Two Tailed Test

Journal: Science Advances

Article Title: PARP1 stabilizes FOXN3 to suppress pulmonary fibrosis through p38-related feedback regulation

doi: 10.1126/sciadv.ady1681

Figure Lengend Snippet: ( A ) Western blot analysis of FOXN3 protein levels in the lungs of Parp1 fl/fl and Parp1 fl/fl Foxn3 LSL/+ mice following BLM treatment (1.5 mg/kg, 21 days) ( n = 4). The mice were treated with or without Cre recombinase. ( B ) Histological trichrome staining assessed lung fibrosis in Parp1 fl/fl and Parp1 fl/fl Foxn3 LSL/+ mice with or without Cre recombinase treatment following BLM treatment (1.5 mg/kg, 21 days). Scale bar, 100 μm. ( C ) The collagen content was quantified using ImageJ software for (B). Four distinct fields for each sample were analyzed and expressed as a percentage of the total area. ( D ) H&E staining analysis was performed to evaluate the inflammatory response in the lungs of Parp1 fl/fl and Parp1 fl/fl Foxn3 LSL/+ mice with or without Cre recombinase treatment following BLM treatment (1.5 mg/kg, 21 days). Scale bar, 100 μm. ( E ) ELISAs were performed to measure the levels of TNFα and IL-6 in BAL fluid from the lungs of Parp1 fl/fl and Parp1 fl/fl Foxn3 LSL/+ mice with or without Cre recombinase treatment following BLM treatment (1.5 mg/kg, 21 days) ( n = 4). ( F ) Quantitative PCR analysis was performed to assess the RNA levels of profibrotic factors in the lungs of Parp1 fl/fl and Parp1 fl/fl Foxn3 LSL/+ mice with or without Cre recombinase treatment following BLM treatment (1.5 mg/kg, 21 days) ( n = 4). ( G ) Hydroxyproline measurements comparing the collagen content in the lungs of Parp1 fl/fl and Parp1 fl/fl Foxn3 LSL/+ mice with or without Cre recombinase treatment following BLM treatment (1.5 mg/kg, 21 days) ( n = 4). ( H ) Anti-ProSPC and anti–α-SMA immunofluorescence staining analysis of lung sections from BLM-challenged (1.5 mg/kg, 21 days) Parp1 fl/fl and Parp1 fl/fl Foxn3 LSL/+ mice with or without Cre recombinase treatment ( n = 4). Fluorescence intensity from four distinct fields for each sample was quantified using ImageJ software and expressed as the relative fluorescence intensity. The data in (D) to (F) were assessed via one-way ANOVA and are shown as the means ± SD. The blotting data for (A) were quantified as the means from three independent experiments using ImageJ software and analyzed using one-way ANOVA. NS means not significant; * P < 0.05 and *** P < 0.001.

Article Snippet: The primary antibodies used for Western blotting were as follows: anti–hemagglutinin (HA) (1:2000, no. 51064-2-AP, Proteintech), anti-Smad4 (1:1000, no. 10231-1-AP, Proteintech), anti-FOXN3 (1:1000, no. 25399-1-AP, Proteintech), anti-Flag (1:1000, no. 20543-1-AP, Proteintech), anti–GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (1:2000, no. 2118, Cell Signaling Technology), anti–α-Tubulin (1:2000, no. 11224-1-AP, Proteintech), anti-FOXN3 (1:1000, no. ab129453, Abcam), anti-PARP1 (1:1000, no. 66520-1-Ig, Proteintech), anti-p38 (1:1000, no. 14064-1-AP, Proteintech), anti–α-SMA (1:500, no. 14395-1-AP, Proteintech), anti-HSP27 (1:500, no. 18284-1-AP, Proteintech), anti–Phospho-HSP27-S78 (1:500, no. 28900-1-AP, Proteintech), anti-FOXN3 (1:500, no. A15039, ABclonal), anti–Cleaved Caspase3 (1:500, no. 9661, Cell Signaling Technology), anti–Cleaved PARP1 (1:500, no. 5625, Cell Signaling Technology), and anti–phospho-FOXN3 S83/85 (1:1000).

Techniques: Western Blot, Staining, Software, Real-time Polymerase Chain Reaction, Immunofluorescence, Fluorescence

Journal: Science Advances

Article Title: PARP1 stabilizes FOXN3 to suppress pulmonary fibrosis through p38-related feedback regulation

doi: 10.1126/sciadv.ady1681

Figure Lengend Snippet: ( A and B ) Western blotting analyzed p38 levels and FOXN3 phosphorylation at S83 and S85 in (A) A549 cells treated with TGF-β (10 ng/ml) over a time course (0, 16, 24, 48 hours) and (B) mouse lungs following BLM administration (1.5 mg/kg) at days 0, 12, and 21. ( C ) Western blotting evaluated p38 levels and FOXN3 phosphorylation in A549 cells treated with or without BYK204165 (20 μM, 4 hours) or PJ34 (5 μM, 4 hours). ( D and E ) Western blot analysis of p38 levels and FOXN3 phosphorylation in A549 cells with or without PARP1 knockdown (D) or KO (E) after TGF-β treatment (10 ng/ml, 16 hours). ( F ) Quantitative PCR detected the RNA levels of p38 in A549 cells treated with or without BYK204165 (20 μM, 4 hours) or PJ34 (5 μM, 4 hours). ( G ) Quantitative PCR detected p38 RNA levels in WT or Parp1 −/− A549 cells treated with TGF-β (10 ng/ml, 16 hours). ( H ) CUT&Tag analyses showing the binding profiles of FOXN3, PARP1, and Smad4 to the p38 promoter in A549 cells treated with or without BYK204165 (20 μM, 4 hours). ( I ) ChIP assays in A549 cells treated with or without BYK204165 (20 μM, 4 hours) assessed FOXN3 and Smad protein binding to the p38 promoter. ( J ) ChIP assays measured FOXN3 and Smad protein binding to the p38 promoter in WT and Parp1 −/− A549 cells after TGF-β treatment (10 ng/ml, 16 hours). ( K ) ChIP assays evaluated Smad protein binding to the p38 promoter in A549 cells with or without FOXN3 knockdown after TGF-β treatment (10 ng/ml, 16 hours). Data from (F), (G), and (I) to (K) were analyzed using two-tailed Student’s t tests and presented as the means ± SD. The blotting data for (A), (C), (D), and (E) were quantified as the means from three independent experiments using ImageJ software. Data for (A), (C), and (D) were analyzed using one-way ANOVA, while data for (E) were assessed using two-tailed Student’s t tests. The blotting data for (B) were also quantified as the means and analyzed by one-way ANOVA. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: The primary antibodies used for Western blotting were as follows: anti–hemagglutinin (HA) (1:2000, no. 51064-2-AP, Proteintech), anti-Smad4 (1:1000, no. 10231-1-AP, Proteintech), anti-FOXN3 (1:1000, no. 25399-1-AP, Proteintech), anti-Flag (1:1000, no. 20543-1-AP, Proteintech), anti–GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (1:2000, no. 2118, Cell Signaling Technology), anti–α-Tubulin (1:2000, no. 11224-1-AP, Proteintech), anti-FOXN3 (1:1000, no. ab129453, Abcam), anti-PARP1 (1:1000, no. 66520-1-Ig, Proteintech), anti-p38 (1:1000, no. 14064-1-AP, Proteintech), anti–α-SMA (1:500, no. 14395-1-AP, Proteintech), anti-HSP27 (1:500, no. 18284-1-AP, Proteintech), anti–Phospho-HSP27-S78 (1:500, no. 28900-1-AP, Proteintech), anti-FOXN3 (1:500, no. A15039, ABclonal), anti–Cleaved Caspase3 (1:500, no. 9661, Cell Signaling Technology), anti–Cleaved PARP1 (1:500, no. 5625, Cell Signaling Technology), and anti–phospho-FOXN3 S83/85 (1:1000).

Techniques: Western Blot, Phospho-proteomics, Knockdown, Real-time Polymerase Chain Reaction, Binding Assay, Protein Binding, Two Tailed Test, Software

Journal: Science Advances

Article Title: PARP1 stabilizes FOXN3 to suppress pulmonary fibrosis through p38-related feedback regulation

doi: 10.1126/sciadv.ady1681

Figure Lengend Snippet: ( A ) Western blotting measured FOXN3 protein levels in the lungs of Parp1 fl/fl and Parp1 fl/fl Foxn3 KI/KI mice following BLM treatment (1.5 mg/kg, 21 days) with or without Cre recombinase administration, as indicated ( n = 3). ( B and C ) Trichrome staining analyzed the fibrotic response in the lungs of Parp1 fl/fl and Parp1 fl/fl Foxn3 KI/KI mice following BLM treatment (1.5 mg/kg, 21 days) with or without Cre recombinase administration (B). Scale bar, 100 μm. Collagen content was quantified using ImageJ (C), with four fields analyzed per sample, expressed as a percentage of total area. ( D ) H&E staining assessed the inflammatory response in the lungs of Parp1 fl/fl and Parp1 fl/fl Foxn3 KI/KI mice following BLM treatment (1.5 mg/kg, 21 days) with or without Cre recombinase administration, as indicated. Scale bar, 100 μm. ( E ) Quantitative PCR assessed the RNA levels of the profibrotic factors in the lungs of Parp1 fl/fl and Parp1 fl/fl Foxn3 KI/KI mice following BLM treatment (1.5 mg/kg, 21 days) with or without Cre recombinase administration, as indicated ( n = 4 for Parp1 fl/fl Foxn3 +/+ Ad-Ctrl, Parp1 fl/fl Foxn3 +/+ Ad-Cre, and Parp1 fl/fl Foxn3 KI/KI Ad-Ctrl; n = 5 for Parp1 fl/fl Foxn3 KI/KI Ad-Cre). ( F ) Hydroxyproline measurements compared collagen content in the lungs of Parp1 fl/fl and Parp1 fl/fl Foxn3 KI/KI mice following BLM treatment (1.5 mg/kg, 21 days) with or without Cre recombinase administration, as indicated ( n = 4 for Parp1 fl/fl Foxn3 +/+ Ad-Ctrl, Parp1 fl/fl Foxn3 +/+ Ad-Cre, and Parp1 fl/fl Foxn3 KI/KI Ad-Ctrl; n = 5 for Parp1 fl/fl Foxn3 KI/KI Ad-Cre). ( G ) ELISAs measured IL-6 and TNFα levels in BAL fluid from the lungs of Parp1 fl/fl and Parp1 fl/fl Foxn3 KI/KI mice following BLM treatment (1.5 mg/kg, 21 days) with or without Cre recombinase administration, as indicated ( n = 4 for Parp1 fl/fl Foxn3 +/+ Ad-Ctrl, Parp1 fl/fl Foxn3 +/+ Ad-Cre, and Parp1 fl/fl Foxn3 KI/KI Ad-Ctrl; n = 5 for Parp1 fl/fl Foxn3 KI/KI Ad-Cre). The data in (C) and (E) to (G) were assessed via one-way ANOVA and are shown as the means ± SD. The blotting data for (A) were quantified as the means from three independent experiments using ImageJ software and analyzed using one-way ANOVA. NS indicates not significant; * P < 0.05 and ** P < 0.01.

Article Snippet: The primary antibodies used for Western blotting were as follows: anti–hemagglutinin (HA) (1:2000, no. 51064-2-AP, Proteintech), anti-Smad4 (1:1000, no. 10231-1-AP, Proteintech), anti-FOXN3 (1:1000, no. 25399-1-AP, Proteintech), anti-Flag (1:1000, no. 20543-1-AP, Proteintech), anti–GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (1:2000, no. 2118, Cell Signaling Technology), anti–α-Tubulin (1:2000, no. 11224-1-AP, Proteintech), anti-FOXN3 (1:1000, no. ab129453, Abcam), anti-PARP1 (1:1000, no. 66520-1-Ig, Proteintech), anti-p38 (1:1000, no. 14064-1-AP, Proteintech), anti–α-SMA (1:500, no. 14395-1-AP, Proteintech), anti-HSP27 (1:500, no. 18284-1-AP, Proteintech), anti–Phospho-HSP27-S78 (1:500, no. 28900-1-AP, Proteintech), anti-FOXN3 (1:500, no. A15039, ABclonal), anti–Cleaved Caspase3 (1:500, no. 9661, Cell Signaling Technology), anti–Cleaved PARP1 (1:500, no. 5625, Cell Signaling Technology), and anti–phospho-FOXN3 S83/85 (1:1000).

Techniques: Western Blot, Staining, Real-time Polymerase Chain Reaction, Software

Journal: Science Advances

Article Title: PARP1 stabilizes FOXN3 to suppress pulmonary fibrosis through p38-related feedback regulation

doi: 10.1126/sciadv.ady1681

Figure Lengend Snippet: ( A and B ) Representative images from the histological immunohistochemical staining analysis showing the expression levels of PARP1 (A) and FOXN3 (B) in the fibrotic lesion areas and the corresponding adjacent normal tissue of patients diagnosed with lung fibrosis. Representative images from four distinct fields for each sample are shown and quantified. Scale bars, 100 μm. ( C ) Results from the quantitative analysis of (A) and (B) are shown as the immunoreactive score (IRS) ( n = 10). ( D ) A proposed working model clarifies the molecular mechanism by which PARP1 regulates the p38-FOXN3 feedback loop to modulate Smad signaling. The schematic representation was created in BioRender. J. Lian (2025); https://biorender.com/p73d358 . The data for (C) was assessed via two-tailed Student’s t tests and are presented as the means ± SD.

Article Snippet: The primary antibodies used for Western blotting were as follows: anti–hemagglutinin (HA) (1:2000, no. 51064-2-AP, Proteintech), anti-Smad4 (1:1000, no. 10231-1-AP, Proteintech), anti-FOXN3 (1:1000, no. 25399-1-AP, Proteintech), anti-Flag (1:1000, no. 20543-1-AP, Proteintech), anti–GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (1:2000, no. 2118, Cell Signaling Technology), anti–α-Tubulin (1:2000, no. 11224-1-AP, Proteintech), anti-FOXN3 (1:1000, no. ab129453, Abcam), anti-PARP1 (1:1000, no. 66520-1-Ig, Proteintech), anti-p38 (1:1000, no. 14064-1-AP, Proteintech), anti–α-SMA (1:500, no. 14395-1-AP, Proteintech), anti-HSP27 (1:500, no. 18284-1-AP, Proteintech), anti–Phospho-HSP27-S78 (1:500, no. 28900-1-AP, Proteintech), anti-FOXN3 (1:500, no. A15039, ABclonal), anti–Cleaved Caspase3 (1:500, no. 9661, Cell Signaling Technology), anti–Cleaved PARP1 (1:500, no. 5625, Cell Signaling Technology), and anti–phospho-FOXN3 S83/85 (1:1000).

Techniques: Immunohistochemical staining, Staining, Expressing, Two Tailed Test