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 PARP1 protein purified from Sf9 insect cells was obtained from MedChemExpress (no. HY- P74652 ).

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 PARP1 protein purified from Sf9 insect cells was obtained from MedChemExpress (no. HY- P74652 ).

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 PARP1 protein purified from Sf9 insect cells was obtained from MedChemExpress (no. HY- P74652 ).

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 PARP1 protein purified from Sf9 insect cells was obtained from MedChemExpress (no. HY- P74652 ).

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 PARP1 protein purified from Sf9 insect cells was obtained from MedChemExpress (no. HY- P74652 ).

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 PARP1 protein purified from Sf9 insect cells was obtained from MedChemExpress (no. HY- P74652 ).

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 PARP1 protein purified from Sf9 insect cells was obtained from MedChemExpress (no. HY- P74652 ).

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

Journal: Molecular cancer research : MCR

Article Title: Let-7 Status is Crucial for PARP1 Expression in HER2-overexpressing Breast Tumors

doi: 10.1158/1541-7786.MCR-16-0287-T

Figure Lengend Snippet: (A, B) Cells were transfected with HER2 or control (CON) siRNA for 72 hours and then analyzed by Western blot analysis for HER2, PARP1, and PARP2 protein levels. β-actin was used as a loading control. MDA-MB-231 cells that (C) transiently or (D) stably express the HER2 vector (231 HER2) as compared to cells expressing the control plasmid (231 NEO) were subjected to Western blot analysis for HER2, PARP1, PARP2, and β-actin. Data shown are representative immmunoblots from one of two independent experiments.

Article Snippet: Luciferase assay The PARP1 3’-UTR clone (SC209859, OriGene Technologies, Rockville, MD, USA) or the vector control (PS100062, OriGene Technologies, Rockville, MD, USA) were co-transfected with the let-7a mimic or let-7a antagomiR along with their respective controls.

Techniques: Transfection, Control, Western Blot, Stable Transfection, Plasmid Preparation, Expressing

Journal: Molecular cancer research : MCR

Article Title: Let-7 Status is Crucial for PARP1 Expression in HER2-overexpressing Breast Tumors

doi: 10.1158/1541-7786.MCR-16-0287-T

Figure Lengend Snippet: Ten nanograms of isolated miRNA from 70% confluent (A) 231 NEO and 231 HER2 or (B) MCF7, BT-474, and SKBR3 human breast cancer cells were reverse transcribed to cDNA and then analyzed by qRT-PCR for let-7a, let-7i, and U6 expression. The figures are representative from (A, B) one of two independent experiments performed in triplicate. A t-test or one-way ANOVA test was performed. (C) Western blot analysis of HER2, PARP1, PARP2 and β-actin levels in untreated 231 NEO, 231 HER2, MCF7, BT-474, and SKBR3 cell lines. **p<0.01, ***p<0.001 and ****p<0.0001.

Article Snippet: Luciferase assay The PARP1 3’-UTR clone (SC209859, OriGene Technologies, Rockville, MD, USA) or the vector control (PS100062, OriGene Technologies, Rockville, MD, USA) were co-transfected with the let-7a mimic or let-7a antagomiR along with their respective controls.

Techniques: Isolation, Reverse Transcription, Quantitative RT-PCR, Expressing, Western Blot

Journal: Molecular cancer research : MCR

Article Title: Let-7 Status is Crucial for PARP1 Expression in HER2-overexpressing Breast Tumors

doi: 10.1158/1541-7786.MCR-16-0287-T

Figure Lengend Snippet: (A–C) BT-474, SKBR3 and 231 HER2 cells were transiently transfected with a let-7a miRNA (hsa-let-7a-5p) or its neg control miRNA (negative control). Following transfection, cells were analyzed by Western blot analysis for PARP-1, PARP-2, and β-actin. (D) The 231 NEO cell line was transiently transfected with an anti-let-7a miRNA (let-7a antagomiR) or a neg control miRNA (negative control). Following transfection, cells were analyzed by Western blot analysis for PARP-1, PARP-2, and β-actin. Results shown are from one of two independent experiments performed duplicate.

Article Snippet: Luciferase assay The PARP1 3’-UTR clone (SC209859, OriGene Technologies, Rockville, MD, USA) or the vector control (PS100062, OriGene Technologies, Rockville, MD, USA) were co-transfected with the let-7a mimic or let-7a antagomiR along with their respective controls.

Techniques: Transfection, Control, Negative Control, Western Blot

Journal: Molecular cancer research : MCR

Article Title: Let-7 Status is Crucial for PARP1 Expression in HER2-overexpressing Breast Tumors

doi: 10.1158/1541-7786.MCR-16-0287-T

Figure Lengend Snippet: (A) The PARP1 3’UTR was aligned with the seed sequence of the let-7a miRNA. (B, C) BT-474 and 231 HER2 cells were co-transfected with a let-7a miRNA or its neg control miRNA and a plasmid containing a firefly luciferase gene and the 3’UTR of PARP1 or a control 3’UTR. (D) 231 NEO cell line was co-transfected with an anti-let-7a miRNA or its neg control miRNA and a plasmid containing a firefly luciferase gene and the 3’UTR of PARP1 or a control 3’UTR. Luciferase activity was normalized to cells co-transfected with the respective neg control miRNA and the plasmid containing the control 3’UTR. The fold change values were calculated back to the cells co-transfected with the respective neg control miRNA and the plasmid containing the 3’UTR of PARP1. Results shown are from one of two independent experiments performed in quadruplicate. *p<0.05, **p<0.01

Article Snippet: Luciferase assay The PARP1 3’-UTR clone (SC209859, OriGene Technologies, Rockville, MD, USA) or the vector control (PS100062, OriGene Technologies, Rockville, MD, USA) were co-transfected with the let-7a mimic or let-7a antagomiR along with their respective controls.

Techniques: Sequencing, Transfection, Control, Plasmid Preparation, Luciferase, Activity Assay

Journal: Molecular cancer research : MCR

Article Title: Let-7 Status is Crucial for PARP1 Expression in HER2-overexpressing Breast Tumors

doi: 10.1158/1541-7786.MCR-16-0287-T

Figure Lengend Snippet: (A) Let-7a levels were measured by qRT-PCR analysis in HER2+ and HER2− breast cancer specimens. U6 was used to normalize data. (B) Let-7a gene and PARP1 protein expression levels were correlated in 17 UAB HER2− and HER2+ breast cancer patients (rs=−0.6937, p=0.0020). ***p<0.001

Article Snippet: Luciferase assay The PARP1 3’-UTR clone (SC209859, OriGene Technologies, Rockville, MD, USA) or the vector control (PS100062, OriGene Technologies, Rockville, MD, USA) were co-transfected with the let-7a mimic or let-7a antagomiR along with their respective controls.

Techniques: Quantitative RT-PCR, Expressing

Journal: International journal of cancer

Article Title: PARP1 expression predicts PARP inhibitor sensitivity and correlates with metastatic potential and overall survival in melanoma.

doi: 10.1002/ijc.34947

Figure Lengend Snippet: FIGURE 1 PARP1 levels are predictive for overall survival and poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) response in melanoma. (A–C) Kaplan Meier curve of overall survival of 459 melanoma patients (A) of the TCGA SKCM 470 dataset divided into stage 0, I, Ia, Ib, IIa, IIb, IIc (B) and stage III, IIIa, IIIb, IIIC, and IV (C) grouped into high and low PARP1 expression. Median was used to define the cutoff between high and low PARP1 expression. R2 was used to plot the graph and perform statistical analysis. (D) 4-Methylumbelliferyl heptanoate (MUH) cell viability assay was performed after treatment of melanoma cells with different concentrations of olaparib or talazoparib for 72 h. IC50 was calculated. PARP1 gene expression normalized to ß-actin of the respective cells is shown. The correlation between the IC50 levels and the PARP1 expression was analyzed using the simple linear regression model. (E) PARPi drug sensitivity using the PRISM Repurposing 19Q4 data compared with PARP1 expression using the DepMap Public 23Q2 is shown. The correlation between talazoparib or olaparib drug sensitivity and the PARP1 expression was analyzed using the simple linear regression model. (F,G) PARP1 gene expression (F) or PARP1 protein expression (G) of PARP1 in six melanocytes, two melanoma cell lines in radial growth phase (RGP), two melanoma cell lines in vertical growth phase (VGP), and six metastatic melanoma cell lines is shown. All experiments were analyzed in four biological replicates. One-way ANOVA with multiple comparison was used to compare the groups. (H) GSE112509 dataset was used to compare PARP1 gene expression between 23 melanocytic nevi and 57 primary melanomas. Unpaired t-test was used to compare the two groups.

Article Snippet: The melanoma cells A375 and SKMel28 were transiently transfected with either siRNA against PARP1 or with a PARP1 expressing plasmid (Origene, RC207085).

Techniques: Expressing, Viability Assay, Gene Expression, Comparison

Journal: International journal of cancer

Article Title: PARP1 expression predicts PARP inhibitor sensitivity and correlates with metastatic potential and overall survival in melanoma.

doi: 10.1002/ijc.34947

Figure Lengend Snippet: FIGURE 2 PARP1 trapping after Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) treatment induces melanoma cell death. (A) Immunoblot analysis of chromatin bound fraction of A375 and SKMel28 cells after the treatment with 0.005% methyl methane sulfonate (MMS) plus 2 μM talazoparib (+) or untreated cells () for 6 h. An exemplary picture of the immunoblot is shown (left side). Relative PARP1 protein expression (ratio PARP1 and H2AX) of three biological replicates is shown in the graph (right side). (B,C) 4-Methylumbelliferyl heptanoate (MUH) cell viability assay of SKMel28 and A375 melanoma cell lines treated with different concentrations of PARPi (olaparib and talazoparib) for 72 h. PARP1 knockdown (siPARP1) and control (Ctr.) cells (B) or PARP1 overexpression (PARP1OvE) and control (Ctr.) (C) transfected cells were used. The treatment started 24 h after the transfection process. Comparison of fits of the nonlinear regression was performed to analyze statistical differences between the untransfected and transfected cells. Relative viability at 25 μM PARPi treatment (ratio of Ctr. vs. siPARP1/ PARP1OvE) of 4 biological replicates (B) and three biological replicates (C) is shown.

Article Snippet: The melanoma cells A375 and SKMel28 were transiently transfected with either siRNA against PARP1 or with a PARP1 expressing plasmid (Origene, RC207085).

Techniques: Western Blot, Expressing, Viability Assay, Knockdown, Control, Over Expression, Transfection, Comparison

Journal: Cell communication and signaling : CCS

Article Title: Poly(ADP-ribosyl)ation of acetyltransferase NAT10 by PARP1 is required for its nucleoplasmic translocation and function in response to DNA damage.

doi: 10.1186/s12964-022-00932-1

Figure Lengend Snippet: Fig. 2 PARP1 PARylates NAT10 at K1016, K1017, and K1020 both in vitro and in vivo. A Four GST-NAT10 deletion mutants (∆1–201, ∆202–488, ∆489–753, and ∆754–1025) and GST control were bacterially purified, and subjected to in vitro PARation assay in the presence of PARP1 and NAD+. The reaction samples were resolved by SDS-PAGE, and analyzed by immunoblotting analyses with anti-PAR and anti-GST antibodies. B In vitro PARylation assays were performed using purified GST-NAT10 deletion fragments in the presence or absence of recombinant PARP1 enzyme, NAD+, and Olaparib. PARylated NAT10 was detected with an anti-PAR antibody. C–E GST-NAT10 deletion fragments were subjected to in vitro PARation assay as described in A. F, G Purified GST-NAT10 754–1025 proteins (WT, K1016A, K1017A, D1018A, and K1020A, K3A) were subjected to in vitro PARation assays in the presence of PARP1 and NAD+. PARylation of NAT10 was detected by immunoblotting with an anti-PAR antibody. In G, K3A represents the combined mutation in all three residues (K1016, K1017, and K1020). H Alignment of the NAT10 protein sequence among different organisms. Asterisk (*) indicates the full conservation of the residues of NAT10 among different species. I, J MCF-7 cells were transfected with HA-NAT10 or HA-NAT10 K3A expression vector. After 48 h of transfection, cells were treated with or without 1 mM MMS for 2 h (I) or 6 Gy IR (J). Thereafter, IP and immunoblotting analyses were conducted with the indicated antibodies

Article Snippet: The purified GST-NAT10 fragment (1 μg) was incubated with 100 ng of recombinant full-length PARP1 (Origene, #TP710053) in a reaction buffer containing 100 mM Tris–HCl (pH 8.0), 10 mM MgCl2, 1 mM DTT, 4 ng/ml sonicated salmon sperm DNA (Invitrogen, #AM9680), and 300 μM nicotinamide adenine dinucleotide (NAD+) at 37 °C for 30 min.

Techniques: In Vitro, In Vivo, Control, Purification, SDS Page, Western Blot, Recombinant, Mutagenesis, Sequencing, Transfection, Expressing, Plasmid Preparation

Journal: Cell communication and signaling : CCS

Article Title: Poly(ADP-ribosyl)ation of acetyltransferase NAT10 by PARP1 is required for its nucleoplasmic translocation and function in response to DNA damage.

doi: 10.1186/s12964-022-00932-1

Figure Lengend Snippet: Fig. 3 PARylation of NAT10 by PARP1 regulates its nucleoplasmic translocation and co-localization with MORC2. A MCF-7 cells were transfected with plasmid DNAs encoding Flag-MORC2, HA-NAT10, or HA-NAT10 K3A. After 48 h of transfection, cells were treated with or without 1 mM MMS for 2 h or 6 Gy IR. IF staining was performed with an anti-Flag (green) or anti-HA (red) antibody. DNA was counterstained with DAPI (blue). Scale bar, 2.5 μm. The quantitative results of cells with NAT10 nucleoplasmic translocation are presented in the right panel. **p < 0.01; NS, no significance. B, C MCF-7 cells were transfected with HA-NAT10 and Flag-MORC2. After 48 h of transfection, cells were pretreated with or without 10 μM Olaparib for 3 h, and then treated with or without 1 mM MMS for another 2 h (B) or 6 Gy IR (C). IF staining was performed with an anti-Flag (green) or anti-HA (red) antibody. DNA was counterstained with DAPI (blue). Scale bar, 2.5 μm. The quantitative results of cells with NAT10 nucleoplasmic translocation are displayed in the right panel. **p < 0.01; ***p < 0.001. D, E PARP1-KO MCF-7 cells were transfected with HA-NAT10 and Flag-MORC2. After 48 h of transfection, cells were treated with or without 1 mM MMS for another 2 h (D) or 6 Gy IR (E). IF staining was performed with an anti-Flag (green) or anti-HA (red) antibody. DNA was counterstained with DAPI (blue). Scale bar, 2.5 μm. The quantitative results of cells with NAT10 nucleoplasmic translocation are displayed in the right panel. **p < 0.01; ***p < 0.001. Arrows indicate the colocalization between MORC2 and NAT10

Article Snippet: The purified GST-NAT10 fragment (1 μg) was incubated with 100 ng of recombinant full-length PARP1 (Origene, #TP710053) in a reaction buffer containing 100 mM Tris–HCl (pH 8.0), 10 mM MgCl2, 1 mM DTT, 4 ng/ml sonicated salmon sperm DNA (Invitrogen, #AM9680), and 300 μM nicotinamide adenine dinucleotide (NAD+) at 37 °C for 30 min.

Techniques: Translocation Assay, Transfection, Plasmid Preparation, Staining

Journal: Cell communication and signaling : CCS

Article Title: Poly(ADP-ribosyl)ation of acetyltransferase NAT10 by PARP1 is required for its nucleoplasmic translocation and function in response to DNA damage.

doi: 10.1186/s12964-022-00932-1

Figure Lengend Snippet: Fig. 4 PARylation of NAT10 by PARP1 regulates its interaction with MORC2. A HEK293T cells were transfected with the indicated expression vectors. After 48 h of transfection, cells were treated with or without 1 mM MMS for 2 h and subjected to IP and immunoblotting analyses with the indicated antibodies. B, C BT549 cells were transfected with plasmid DNAs encoding pCDH, HA-NAT10, or HA-NAT10 K3A. After 48 h of transfection, cells were treated with or without 1 mM MMS for 2 h (B) or 6 Gy IR (C). IP and immunoblotting analyses were performed with the indicated antibodies. D–F MCF-7 and BT549 cells were pretreated with or without 10 μM Olaparib for 3 h, and then treated with or without 1 mM MMS for another 2 h. The sequential IP and immunoblotting analyses were performed with the indicated antibodies. G–I MCF-7 and BT549 cells were pretreated with or without 10 μM Olaparib for 3 h, and then treated with or without 6 Gy IR. The sequential IP and immunoblotting analyses were performed with the indicated antibodies

Article Snippet: The purified GST-NAT10 fragment (1 μg) was incubated with 100 ng of recombinant full-length PARP1 (Origene, #TP710053) in a reaction buffer containing 100 mM Tris–HCl (pH 8.0), 10 mM MgCl2, 1 mM DTT, 4 ng/ml sonicated salmon sperm DNA (Invitrogen, #AM9680), and 300 μM nicotinamide adenine dinucleotide (NAD+) at 37 °C for 30 min.

Techniques: Transfection, Expressing, Western Blot, Plasmid Preparation

Journal: Cell communication and signaling : CCS

Article Title: Poly(ADP-ribosyl)ation of acetyltransferase NAT10 by PARP1 is required for its nucleoplasmic translocation and function in response to DNA damage.

doi: 10.1186/s12964-022-00932-1

Figure Lengend Snippet: Fig. 6 DNA damage induces MORC2 K767Ac in a PARP1-dependent manner. A MCF-7 cells were pretreated with or without 10 μM ATM inhibitor (KU-55933), 10 μM ATR inhibitor (VE-821), 10 μM DNA-PKcs inhibitor (NU7441), and 10 μM PARP inhibitor (Olaparib) for 3 h, and then treated with 1 mM MMS for 1 h. IP and immunoblotting analyses were performed with the indicated antibodies. Positive controls for these inhibitors are shown in the input. B HEK293T cells stably expressing pCDH and Flag-MORC2 were pretreated with or without 10 μM Olaparib for 3 h, and then treated with 1 mM MMS for another 2 h or 6 Gy IR. IP and immunoblotting analyses were performed with the indicated antibodies. C MCF-7 and BT549 cells were pretreated with or without 10 μM Olaparib for 3 h, and then treated with 1 mM MMS for another 2 h or 6 Gy IR. IP and immunoblotting analyses were performed with the indicated antibodies. D WT and PARP1-KO MCF-7 cells were treated with or without 1 mM MMS for 2 h or 6 Gy IR. Thereafter, IP and immunoblotting analyses were carried out with the inidicated antibodies. E BT549 cells were transfected with siNC or two independent siRNA targeting PARP1 (siPARP1). After 48 h of transfection, cells were treated with or without 1 mM MMS for 2 h or 6 Gy IR. IP and immunoblotting analyses were subsequently performed with the indicated antibodies

Article Snippet: The purified GST-NAT10 fragment (1 μg) was incubated with 100 ng of recombinant full-length PARP1 (Origene, #TP710053) in a reaction buffer containing 100 mM Tris–HCl (pH 8.0), 10 mM MgCl2, 1 mM DTT, 4 ng/ml sonicated salmon sperm DNA (Invitrogen, #AM9680), and 300 μM nicotinamide adenine dinucleotide (NAD+) at 37 °C for 30 min.

Techniques: Western Blot, Stable Transfection, Expressing, Transfection

Journal: Cell communication and signaling : CCS

Article Title: Poly(ADP-ribosyl)ation of acetyltransferase NAT10 by PARP1 is required for its nucleoplasmic translocation and function in response to DNA damage.

doi: 10.1186/s12964-022-00932-1

Figure Lengend Snippet: Fig. 5 PARylation of NAT10 by PARP1 regulates MORC2 acetylation in response to DNA damage. A, B NAT10-KO MCF-7 and BT549 cells were transfected with plasmid DNAs encoding pCDH, HA-NAT10, or HA-NAT10 K3A. After 48 h of transfection, cells were treated with or without 1 mM MMS for 2 h (A) or 6 Gy IR (B), and then subjected to IP and immunoblotting with the indicated antibodies

Article Snippet: The purified GST-NAT10 fragment (1 μg) was incubated with 100 ng of recombinant full-length PARP1 (Origene, #TP710053) in a reaction buffer containing 100 mM Tris–HCl (pH 8.0), 10 mM MgCl2, 1 mM DTT, 4 ng/ml sonicated salmon sperm DNA (Invitrogen, #AM9680), and 300 μM nicotinamide adenine dinucleotide (NAD+) at 37 °C for 30 min.

Techniques: Transfection, Plasmid Preparation, Western Blot

Journal: Cell communication and signaling : CCS

Article Title: Poly(ADP-ribosyl)ation of acetyltransferase NAT10 by PARP1 is required for its nucleoplasmic translocation and function in response to DNA damage.

doi: 10.1186/s12964-022-00932-1

Figure Lengend Snippet: Fig. 7 PARylation of NAT10 by PARP1 is required for cell survival in response to DNA damage. A, B NAT10-KO MCF-7 and BT549 cells stably expressing pCDH, HA-NAT10 WT, or HA-NAT10 K3A were treated with increasing doses of MMS and then subjected to clonogenic survival assays. Representative images of survival colonies are displayed in A and the corresponding quantitative results are shown in B. C, D NAT10-KO MCF-7 and BT549 cells stably expressing pCDH, HA-NAT10 WT, or HA-NAT10 K3A were treated with or without 6 Gy IR, and then subjected to clonogenic survival assays. Representative images of survival colonies are displayed in C and the corresponding quantitative results are shown in D

Article Snippet: The purified GST-NAT10 fragment (1 μg) was incubated with 100 ng of recombinant full-length PARP1 (Origene, #TP710053) in a reaction buffer containing 100 mM Tris–HCl (pH 8.0), 10 mM MgCl2, 1 mM DTT, 4 ng/ml sonicated salmon sperm DNA (Invitrogen, #AM9680), and 300 μM nicotinamide adenine dinucleotide (NAD+) at 37 °C for 30 min.

Techniques: Stable Transfection, Expressing

Journal: Cell communication and signaling : CCS

Article Title: Poly(ADP-ribosyl)ation of acetyltransferase NAT10 by PARP1 is required for its nucleoplasmic translocation and function in response to DNA damage.

doi: 10.1186/s12964-022-00932-1

Figure Lengend Snippet: Fig. 8 The proposed working model. Activated PARP1 after DNA damage catalyzes the PARylation of NAT10, which is required for the translocation of NAT10 from the nucleolus to the nucleoplasm. NAT10 relocalization increases its co-localization and interaction with its substrate, MORC2, thereby enhancing MORC2 K767Ac in response to DNA damage

Article Snippet: The purified GST-NAT10 fragment (1 μg) was incubated with 100 ng of recombinant full-length PARP1 (Origene, #TP710053) in a reaction buffer containing 100 mM Tris–HCl (pH 8.0), 10 mM MgCl2, 1 mM DTT, 4 ng/ml sonicated salmon sperm DNA (Invitrogen, #AM9680), and 300 μM nicotinamide adenine dinucleotide (NAD+) at 37 °C for 30 min.

Techniques: Translocation Assay

Journal: Science Advances

Article Title: USP1 deubiquitinates PARP1 to regulate its trapping and PARylation activity

doi: 10.1126/sciadv.adp6567

Figure Lengend Snippet: ( A ) Graphs representing cell viability of COV-318 and OVCAR-8 cells after 16 hours of treatment with the specific PARP1 inhibitor AZD-9574, SJB, or the combination (COV-318: SJB 200 nM and AZD 500 μM; OVCAR-8: SJB 100 nM and AZD 250 μM). Data are expressed as percentage of viable cells with respect to the untreated conditions and represented the mean (±SD). ( B and C ) Coimmunoprecipitation (co-IP) analysis of exogenous in 293T17 (B) or endogenous in COV-318 cells (C) interaction between USP1 and PARP1 proteins. ( D ) Co-IP analysis of endogenous USP1 and PARP1 interaction in OVCAR-8 cells treated or not with SJB (500 nM) or niraparib (1μM) for 3 hours. ( E ) Representative confocal microscopy images of endogenous PARP1/USP1 proximity ligation assay (PLA) in OVCAR-8 cells treated or not with SJB (500 nM) for 3 hours. White dashed boxes highlight the areas magnified in the panels below. The graph on the right report the fluorescence intensity/cells for each condition (>60 cells analyzed per condition, n = 3). ( F ) Schematic representation of PARP1-truncated mutants used in this work. ( G ) Co-IP analysis performed in whole lysates of 293T17 cells transfected with FLAG-tagged plasmids for PARP1 described in (F) together with plasmid encoding GFP-tagged USP1 (* = aspecific band). In (B), (C), (D), and (G), input indicates the expression of PARP1 and USP1 in cell lysates. Immunoglobulin G (IgG) represents the control IP using an unrelated antibody. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or tubulin was used as loading controls. In (A) and (E), two-tailed, unpaired Student’s t test was used (*** P < 0.001 and **** P < 0.0001).

Article Snippet: Human GFP-tagged PARP1 was provided from OriGene (no. RG207085). pCI-His-hUbi was from Astar Winoto (provided from Addgene no. 31815).

Techniques: Co-Immunoprecipitation Assay, Confocal Microscopy, Proximity Ligation Assay, Fluorescence, Transfection, Plasmid Preparation, Expressing, Control, Two Tailed Test

Journal: Science Advances

Article Title: USP1 deubiquitinates PARP1 to regulate its trapping and PARylation activity

doi: 10.1126/sciadv.adp6567

Figure Lengend Snippet: ( A ) His-Ubiquitin pull down assay of Flag-PARP1 in 293T17 cells co-transfected with His-tagged ubiquitin, PARP1, USP1 WT , or USP1 C90S . ( B ) Endogenous PARP1 ubiquitination analysis in OVCAR-8 USP1 WT or KO cells transfected with His-tagged ubiquitin. Whole-cell lysates were subjected to IP with antibody against PARP1 and immunoblotted for specific K63-linked ubiquitin chains. ( C ) Co-IP analysis on 293T17 cells lysates transfected with His-tagged ubiquitin and treated with the inhibitors as indicated for 3 hours (SJB 800 nM and Nir./ML-792/MLN-7243 1 μM). α-PARP1 antibody was used for the immunoprecipitation. ( D ) Nonlinear regression analysis of cell viability assay in OVCAR-8 cells transfected with Flag-tagged PARP1 full-length (FL) or ΔBRCT/WGR deletion mutant and treated with increasing doses of niraparib together or not with SJB (50 nM). Overexpression was checked by WB analysis. Data and statistical analysis were calculated and expressed as described in . The table below the graph shows the IC50 and the CI of each condition. In (A) to (C), input indicates the expression of PARP1, USP1, and ubiquitin in cell lysates. IgG represents the control IP using an unrelated antibody. GAPDH or tubulin was used as loading controls.

Article Snippet: Human GFP-tagged PARP1 was provided from OriGene (no. RG207085). pCI-His-hUbi was from Astar Winoto (provided from Addgene no. 31815).

Techniques: Pull Down Assay, Transfection, Co-Immunoprecipitation Assay, Immunoprecipitation, Viability Assay, Mutagenesis, Over Expression, Expressing, Control

Journal: Science Advances

Article Title: USP1 deubiquitinates PARP1 to regulate its trapping and PARylation activity

doi: 10.1126/sciadv.adp6567

Figure Lengend Snippet: ( A and B ) WB analysis of PAR content in EOC cells treated with SJB (500 nM for OVCAR-8 and 300 nM for COV-318) for 3 hours (A) or in COV-318 as indicated (B). ( C ) PARP1 activity analyzed by ELISA in COV-318 cells treated with SJB (300 nM) or niraparib (Nir) (1 μM) for 16 hours. Results are expressed as mUnits PARP/ng protein (mean ± SD of three biological determinations). ( D ) WB analysis of PAR content in whole lysates of OVCAR-8 USP1 WT or USP1 KO cells treated or not with niraparib as indicated for 24 hours. ( E ) Co-IP analysis of endogenous PARP1 auto-PARylation in OVCAR-8 cells treated with SJB (500 nM) or niraparib (1 μM) for 3 hours. ( F ) WB analysis of USP1 and PARP1 trapping in whole lysates and chromatin fractions of COV-318 cells treated with SJB (300 nM) as indicated. ( G ) Representative images showing GFP-PARP1 accumulation at laser-induced DNA damage (dashed boxes) at the indicated time points in OVCAR-8 cells treated or not with SJB (500 nM, 3 hours). ( H and I ) Graphs representing the percentage of recovery of the damaged area over time (H) and at last recorded frame (4 min) (I) (>40 cells were considered/condition). Two-tailed unpaired t test with Welch’s correction was used (*** P < 0.001). ( J and L ) Co-IP analysis of endogenous USP1 and PARP1 interaction (J) and PARP1 polyubiquitination (L) on chromatin of OVCAR-8 cells treated with SJB (500 nM) for 3 hours. (* = aspecific band). ( K ) Representative confocal microscopy images of endogenous PARP1 and ubiquitin (Ubi) PLA in OVCAR-8 cells overexpressing His-ubiquitin and treated or not with SJB (500 nM, 3 hours). White dashed boxes highlight the magnified areas. The graphs on the right report the fluorescence intensity/cells for each condition (>100 cells analyzed, n = 3). ( M ) WB analysis of PARP1 expression on whole lysates and chromatin fractions of OVCAR-8 overexpressing Flag-PARP1 FL or ΔBRCT/WGR mutant and treated as in (J). In (C) and (K), two-tailed, unpaired Student’s t test was used (* P < 0.05 and **** P < 0.0001)

Article Snippet: Human GFP-tagged PARP1 was provided from OriGene (no. RG207085). pCI-His-hUbi was from Astar Winoto (provided from Addgene no. 31815).

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Co-Immunoprecipitation Assay, Two Tailed Test, Confocal Microscopy, Fluorescence, Expressing, Mutagenesis

Journal: Science Advances

Article Title: USP1 deubiquitinates PARP1 to regulate its trapping and PARylation activity

doi: 10.1126/sciadv.adp6567

Figure Lengend Snippet: ( A and B ) iPOND analyses of endogenous PARP1 (A) and exogenous USP1 (B) at replication forks in OVCAR-8 cells transfected (B) or not (A) with GFP-tagged USP1 and treated with SJB (500 nM) for 3 hours. Inputs represent 2% of the total cellular protein content. ( C ) Representative confocal microscopy images of SIRF analysis in OVCAR-8 cells treated with SJB (500 nM) for 3 hours. Green dots represent PARP1/biotin PLA foci. ( D ) Representative confocal microscopy images of PLA analysis between USP1-γH2AX in COV-318 cells treated or not with SJB (300 nM) for 3 hours. ( E to H ) WB analysis of USP1 and PARP1 (F) or RS markers (H) expression or images from PARP1/γH2AX PLA (G) (>150 cells counted in each condition, n = 3) in OVCAR-8 cells used for a trapping–chase experiment according to the experiments scheme reported in (E) (500 nM SJB, 1 μM Nir). In (C), (D), and (G), the graphs report the fluorescence intensity/cells for each condition expressed with mean ± SD of quantifications of green dots for each cell. Two-tailed, unpaired Student’s t test was used (* P < 0.05 and **** P < 0.0001). ( I ) Schematic representation of the proposed USP1-mediated processing of PARP1 deubiquitination. Created with BioRender.com. In (A) and (B), Input indicates the expression of PARP1 and USP1 in cell lysates. IgG represents the control IP using an unrelated antibody. GAPDH was used as loading control for whole lysates, Histone H3 for chromatin fractions.

Article Snippet: Human GFP-tagged PARP1 was provided from OriGene (no. RG207085). pCI-His-hUbi was from Astar Winoto (provided from Addgene no. 31815).

Techniques: Transfection, Confocal Microscopy, Expressing, Fluorescence, Two Tailed Test, Control