Journal: Experimental & Molecular Medicine

Article Title: Poly(ADP-ribose) polymerase 1 orchestrates vascular smooth muscle cell homeostasis in arterial disease

doi: 10.1038/s12276-025-01501-5

Figure Lengend Snippet: a , Double immunofluorescence staining for poly(ADP-ribose) polymer (PAR, green) and VSMC marker αSMA (red) in SOCs and balloon-injured rat carotid arteries after injury ( n = 5–8 for each time point). Nuclei were stained with DAPI (blue). b , Immunoblot assay of poly(ADP-ribosyl)ated proteins in lysates of SOCs and balloon-injured arteries at various time points after injury. c , PARP1 was immunoprecipitated from lysates of SOCs and balloon-injured arteries, followed by immunoblot with anti-PAR antibody (top); PARP1 expression in the lysates was determined using immunoblot (bottom) ( n = 5). d , Double immunofluorescence staining for PAR (red) and αSMA (green) in human samples of normal internal mammary artery ( n = 6), coronary artery after stenting ( n = 4), coronary atherosclerotic plaque ( n = 8) and transplant renal artery stenosis ( n = 4). e , PARP1 was immunoprecipitated from lysates of human internal mammary artery and coronary artery after stenting ( n = 4, left) or atherosclerotic plaque ( n = 6, right), followed by immunoblot with anti-PAR antibody. f , g , Reanalysis of previously reported single-cell RNA-seq dataset generated from balloon-injured carotid arteries of rats ( GSE174098 ) showing that a majority of genes involved in cell proliferation and migration were positively correlated with PARP1 expression, and those pathways related to VSMC contraction were negatively associated with mRNA level of PARP1. f shows the Circos heatmap of genes associated with PARP1, and g provides a detailed view of the genes involved in cell contraction, proliferation, and migration. Scale bar, 50 μm. Throughout, data are presented as mean ± s.d. * P < 0.05.

Article Snippet: In brief, 300 μg of protein extracts were incubated with the indicated antibodies against PARP1, PAR, myocardin (Sigma-Aldrich, SAB4200539), SRF (Cell Signaling Technology, 5147), FLAG (Cell Signaling Technology, 14793), His (Cell Signaling Technology, 12698), HA (Cell Signaling Technology, 3724), c-Jun (Cell Signaling Technology, 9165), p65 (Cell Signaling Technology, 8242) or unspecific IgG at 4 °C overnight, and protein-A/G agarose was added for another 2–3 h at 4 °C.

Techniques: Double Immunofluorescence Staining, Polymer, Marker, Staining, Western Blot, Immunoprecipitation, Expressing, RNA Sequencing, Generated, Migration

Journal: Experimental & Molecular Medicine

Article Title: Poly(ADP-ribose) polymerase 1 orchestrates vascular smooth muscle cell homeostasis in arterial disease

doi: 10.1038/s12276-025-01501-5

Figure Lengend Snippet: a , PARP1-containing protein complexes were immunoaffinity purified from T/G-HAVSMC nuclear extracts and then subjected to SDS–PAGE and analyzed by LC–MS/MS assay. Black letters indicate the peptides identified. b , Co-IP assay of PARP1-bound proteins and myocardin-bound proteins in nuclear extracts from T/G-HAVSMCs infected with Flag-myocardin adenovirus, followed by immunoblot with anti-Flag antibody (top) and with anti-PARP1 antibody (bottom) ( n = 3). Nonspecific IgG served as a negative control. c , IP of myocardin and poly(ADP-ribosyl)ated proteins in nuclear extracts from T/G-HAVSMCs infected with Flag-myocardin adenovirus, followed by immunoblot with anti-PAR antibody (top) and with anti-Flag antibody (bottom) ( n = 3). Nonspecific IgG served as a negative control. d , Poly(ADP-ribosyl)ation of myocardin immunoprecipitated from nuclear extracts of mVSMCs infected with Flag-myocardin adenovirus, which were preincubated with active DNA/NAD + alone or together with recombinant human PARP1 protein (hPARP1), was determined using immunoblot. e , Poly(ADP-ribosyl)ation of myocardin immunoprecipitated from nuclear extracts of T/G-HAVSMCs transfected with different mutant constructs, which were preincubated with active DNA/NAD + alone or together with recombinant human PARP1 protein (hPARP1), was determined using immunoblot. f , Poly(ADP-ribosyl)ation of myocardin in nuclear extracts from VSMCs transfected with WT or mutant myocardin plasmid, which were incubated with active DNA/NAD + alone or together with hPARP1 or 3AB, was determined using immunoblot. g , Balloon-injured rat arteries were in-site infected with Ad-Scr shRNA, Ad-PARP1 shRNA or adenoviruses encoding myocardin shRNA (Ad-myocardin shRNA) immediately after injury and then were treated with PJ34 for 10 days ( n = 5–8). mRNA levels of Parp1 , myocardin , αSMA , Sm22α , CyclinD1 , Pcna, Mmp2 and Mmp9 in injured arteries at 10 days. h , i , rVMSCs were infected with Ad-myocardin shRNA or Ad-Scr shRNA and then treated with PDGF-BB + PJ34 for 48 h. The gene transcription profiles were analyzed by RNA-seq: heat map of DEGs ( h ); genes related to VSMC contraction, proliferation and migration were separated ( i ). Throughout, data are presented as mean ± s.d. * P < 0.05 versus injury + Scr shRNA or injury + myocardin shRNA.

Article Snippet: In brief, 300 μg of protein extracts were incubated with the indicated antibodies against PARP1, PAR, myocardin (Sigma-Aldrich, SAB4200539), SRF (Cell Signaling Technology, 5147), FLAG (Cell Signaling Technology, 14793), His (Cell Signaling Technology, 12698), HA (Cell Signaling Technology, 3724), c-Jun (Cell Signaling Technology, 9165), p65 (Cell Signaling Technology, 8242) or unspecific IgG at 4 °C overnight, and protein-A/G agarose was added for another 2–3 h at 4 °C.

Techniques: Purification, SDS Page, Liquid Chromatography with Mass Spectroscopy, Co-Immunoprecipitation Assay, Infection, Western Blot, Negative Control, Immunoprecipitation, Recombinant, Transfection, Mutagenesis, Construct, Plasmid Preparation, Incubation, shRNA, RNA Sequencing, Migration

Journal: Experimental & Molecular Medicine

Article Title: Poly(ADP-ribose) polymerase 1 orchestrates vascular smooth muscle cell homeostasis in arterial disease

doi: 10.1038/s12276-025-01501-5

Figure Lengend Snippet: a , The interaction among His-tagged PARP1, Flag-tagged myocardin and HA-tagged SRF in 293T cells was determined using Co-IP assay. b , A7r5 cells were infected with Ad-myocardin shRNA or Ad-SRF shRNA. PARP1-bound SRF and myocardin was detected using co-IP assays ( n = 3). c , Poly(ADP-ribosyl)ation of SRF immune- precipitated from T/G-HAVSMC nuclear extracts (top) or aortic lysates of WT and PKO mouse (bottom) was determined using immunoblot. d , Poly(ADP-ribosyl)ation of SRF in nuclear extracts from WT-mVSMCs or PKO-mVSMCs, which were incubated with active DNA/NAD + alone or together with hPARP1, was determined using immunoblot. e , A7r5 cells infected with Flag-myocardin adenovirus, and exposed to PDGF-BB treatment for 48 h. Chromatin was first immunoprecipitated with anti-SRF antibody, and then re-immunoprecipitated with anti-PAR, anti-PARP1, anti-Flag (myocardin) or anti-IgG antibody. f , ChIP assay using anti-myocardin (left) or anti-SRF (right) for amplification of rat SM22α promoters in rVSMCs treated with PARP inhibitors (3AB, PJ34) or infected with Ad-PARP1 shRNA in the presence of PDGF-BB for 48 h. g , Luciferase reporters driven by WT or CArG-mutant SM22α promoter (WT- SM22α and MUT- SM22α , respectively) or 6×CArG were transfected into PDGF-BB-treated WT- or PKO-mVSMCs, and luciferase activities were thereafter analyzed ( n = 4). h , TF target gene sets were collected from the MSigDB C3 TFT motif gene sets, and multiple target gene sets of certain TF were combined to serve as an integrated targets source, including SRF_01, SRF_C, SRF_Q4, SRF_Q5_01 and SRF_Q6. Fisher’s exact test was used to detect TF targets overrepresentation in DEGs. i , A7r5 cells were preinfected with Ad-PARP1, Ad-myocardin and Ad-SRF as indicated, and the luciferase activities driven by WT-SM22α (left), MUT-SM22α (middle) or 6×CArG (right) were determined at 48 h after reporter transfection ( n = 4). Throughout, data are presented as mean ± s.d. * P < 0.05 versus control or WT or Vector, # P < 0.05 versus P DGF-BB or PDGF-BB + Scr shRNA or Ad-myocardin or Ad-myocardin + Ad-SRF.

Article Snippet: In brief, 300 μg of protein extracts were incubated with the indicated antibodies against PARP1, PAR, myocardin (Sigma-Aldrich, SAB4200539), SRF (Cell Signaling Technology, 5147), FLAG (Cell Signaling Technology, 14793), His (Cell Signaling Technology, 12698), HA (Cell Signaling Technology, 3724), c-Jun (Cell Signaling Technology, 9165), p65 (Cell Signaling Technology, 8242) or unspecific IgG at 4 °C overnight, and protein-A/G agarose was added for another 2–3 h at 4 °C.

Techniques: Co-Immunoprecipitation Assay, Infection, shRNA, Western Blot, Incubation, Immunoprecipitation, Amplification, Luciferase, Mutagenesis, Transfection, Control, Plasmid Preparation

Journal: Experimental & Molecular Medicine

Article Title: Poly(ADP-ribose) polymerase 1 orchestrates vascular smooth muscle cell homeostasis in arterial disease

doi: 10.1038/s12276-025-01501-5

Figure Lengend Snippet: a , Luciferase activity driven by 6×AP1 reporter in A7r5 cells, which were infected with Ad-PARP1 or Ad-mut-PARP1 (enzymatic mutant) ( n = 4). b , TF target gene sets were collected from the MSigDB C3 TFT motif gene sets, and multiple target gene sets of certain TF were combined to serve as an integrated targets source, including AP1: AP_1, AP_C, AP1_Q2, AP1_Q2_01, AP1_Q4, AP1_Q4_01, AP1_Q6, AP1_Q6_01, P65: NFKAPPAB65_01. Fisher’s exact test had been used to detect TF target overrepresentation in DEGs of vehicle, PDGF-BB and PDGF-BB + PJ34. c , d , Luciferase activity driven by WT or AP1-binding site mutant CyclinD1 promoter (WT- CyclinD1 and MUT- CyclinD1 , respectively) ( c ), or 6×AP1 ( d ) in A7r5 cells infected with Ad-myocardin or Ad-Null ( n = 4). e , TF target gene sets were collected from the MSigDB C3 TFT motif gene sets, and multiple target gene sets of certain TF were combined to serve as an integrated targets source, including AP1: AP_1, AP_C, AP1_Q2, AP1_Q2_01, AP1_Q4, AP1_Q4_01, AP1_Q6, AP1_Q6_01, P65: NFKAPPAB65_01. Fisher’s exact test had been used to detect TF target overrepresentation in DEGs of PDGF-BB + PJ34 + Scr and PDGF-BB + PJ34 + myocardin shRNA. f , g , A7r5 cells were infected with Ad-myocardin or Ad-PARP1, and the luciferase activity driven by WT- CyclinD1 (left), MUT- CyclinD1 (right) ( f ) or 6×AP1 ( g ) in A7r5 cells were determined at 48 h after reporter transfection ( n = 4). h , Nuclear extracts from T/G-HAVSMCs infected with Flag-myocardin adenovirus were incubated with active DNA and NAD + (0, 1, 10 or 200 μM). Coprecipitates of myocardin with c-Jun or p65, and coprecipitates of PARP1 with c-Jun or p65, were determined using co-IP assay. i , Coprecipitates of c-Jun and myocardin in nuclear extracts from Ad-PARP1 or Ad-mut-PARP1-infected T/G-HAVSMCs along with Flag-myocardin adenoviral transduction ( n = 4). j , Coprecipitates of c-Jun and myocardin in nuclear extracts from Flag-myocardin-infected A7r5 cells infected with Ad-Scr shRNA or Ad-PARP1 shRNA (left); coprecipitates and poly(ADP-ribosyl)ation of c-Jun and myocardin in nuclear extracts from PDGF-BB-treated rVSMCs in the presence or absence of PJ34. k , A7r5 cells infected with Flag-myocardin adenovirus were treated with vehicle or PDGF-BB for 48 h. Chromatin was first immunoprecipitated with anti-c-Jun antibody, and then re-immunoprecipitated with anti-PAR, anti-PARP1, anti-Flag (myocardin) or anti-IgG antibody, respectively. Throughout, data are presented as mean ± s.d. * P < 0.05 versus Ad-Null, # P < 0.05 versus Ad-PARP1.

Article Snippet: In brief, 300 μg of protein extracts were incubated with the indicated antibodies against PARP1, PAR, myocardin (Sigma-Aldrich, SAB4200539), SRF (Cell Signaling Technology, 5147), FLAG (Cell Signaling Technology, 14793), His (Cell Signaling Technology, 12698), HA (Cell Signaling Technology, 3724), c-Jun (Cell Signaling Technology, 9165), p65 (Cell Signaling Technology, 8242) or unspecific IgG at 4 °C overnight, and protein-A/G agarose was added for another 2–3 h at 4 °C.

Techniques: Luciferase, Activity Assay, Infection, Mutagenesis, Binding Assay, shRNA, Transfection, Incubation, Co-Immunoprecipitation Assay, Transduction, Immunoprecipitation

Journal: Journal of leukocyte biology

Article Title: Expression of and functional responses to protease-activated receptors on human eosinophils.

doi: 10.1189/jlb.0702351

Figure Lengend Snippet: Fig. 1. RT-PCR analysis of human leu- kocytes and endothelial cells for PAR RNA expression. Total RNA was ex- tracted from human eosinophils (A), neu- trophils (B), mononuclear cells (C), or en- dothelial cells (HUVECs; D) and was sub- ject to RT-PCR using random hexamer primers for the RT step and PAR-specific primers for the PCR step. PCR products were analyzed by agarose gel electro- phoresis and compared with a 100-bp lad- der (left lane). Expected product sizes were: PAR1, 300 bp; PAR2, 399 bp; PAR3, 372 bp; PAR4, 242 bp. Figure is a representative example of five different donors.

Article Snippet: Mouse monoclonal antibodies (mAb) to the N-terminal of PAR1 [ATAP2, mouse immunoglobulin G1 (IgG1)] and PAR2 (SAM11, mouse IgG2a) and goat polyclonal antibodies (pAb) directed against the C terminus of each of the PARs were purchased from Santa Cruz (Autogen Bioclear, Wiltshire, UK).

Techniques: Reverse Transcription Polymerase Chain Reaction, RNA Expression, Random Hexamer, Agarose Gel Electrophoresis

Journal: Journal of leukocyte biology

Article Title: Expression of and functional responses to protease-activated receptors on human eosinophils.

doi: 10.1189/jlb.0702351

Figure Lengend Snippet: Fig. 3. Flow cytometry of human eosinophils for PAR1 and PAR2. Purified human eosinophils were stained with mAb to PAR1 (ATAP2, A and B) or PAR2 (SAM11, C and D). Cells were unfixed to study surface-only expression (A and C) or fixed and permeabilized to look at surface and intracellular levels (B and D). mAb are shown by the solid line, and appropriate isotype controls are represented by the dotted lines. (A–D) Representative example of four independent experiments. The levels of PAR1 and PAR2 in purified eosinophils or mixed granulocyte population following fixation and permeabilization were measured in control and asthmatic donors (each group, n4). (E) The results are shown as a ratio of the mean fluorescence intensity (MFI) of PAR mAb:isotype control.

Article Snippet: Mouse monoclonal antibodies (mAb) to the N-terminal of PAR1 [ATAP2, mouse immunoglobulin G1 (IgG1)] and PAR2 (SAM11, mouse IgG2a) and goat polyclonal antibodies (pAb) directed against the C terminus of each of the PARs were purchased from Santa Cruz (Autogen Bioclear, Wiltshire, UK).

Techniques: Flow Cytometry, Staining, Expressing, Control

Journal: Journal of leukocyte biology

Article Title: Expression of and functional responses to protease-activated receptors on human eosinophils.

doi: 10.1189/jlb.0702351

Figure Lengend Snippet: Fig. 5. GAFS assay to measure shape change in response to PAR agonists in eosinophils (Eos) and neutrophils (Neuts). The GAFS assay was used to assess shape change by measuring increases in FSc of a mixed population of eosinophils and neu- trophils using flow cytometry. The eosinophil pop- ulation (R1) is distinguished from the neutrophils (R2) by their autofluorescent properties on the FL2 channel (A). Resting eosinophils and neutrophils (C and D) showed an increase in mean FSc when stimulated with PAF (E, P0.001; F, P0.001). (B) The mean FSc of resting and stimulated cells from a typical experiment is shown. Eotaxin was also used as a control to increase the mean FSc in eosinophils only. (G and H) The results shown are expressed as the percent increase in FSc induced by each agonist compared with buffer-stimulated cells after 5 and 30 min incubation (n6). Eosin- ophils (G) responded to the PAR1 peptide at both time points (5 min, P0.004; 30 min, P0.010) but not to thrombin. Activation via trypsin or PAR2 peptide (P0.008) was only seen after 30 min. Neutrophils (H) responded to trypsin after 30 min (P0.033) and at 5 and 30 min with peptides to PAR1 (5 min, P0.023; 30 min, P0.009) and PAR2 (5 min, P0.021; 30 min, P0.001). PAR4 was considered ineffective in this assay in either cell type.

Article Snippet: Mouse monoclonal antibodies (mAb) to the N-terminal of PAR1 [ATAP2, mouse immunoglobulin G1 (IgG1)] and PAR2 (SAM11, mouse IgG2a) and goat polyclonal antibodies (pAb) directed against the C terminus of each of the PARs were purchased from Santa Cruz (Autogen Bioclear, Wiltshire, UK).

Techniques: Cytometry, Control, Incubation, Activation Assay

Journal: Journal of leukocyte biology

Article Title: Expression of and functional responses to protease-activated receptors on human eosinophils.

doi: 10.1189/jlb.0702351

Figure Lengend Snippet: Fig. 6. Release of cys-LT from human eosinophils in response to PAR activation. Purified human eosinophils were primed with 5 ng/ml IL-5 and treated with PAR agonists for 60 min. Cell-free supernatants were assayed for cys-LT using a commercially available EIA (n4–7). Release of cys-LT was very donor-variable, and only PAR2 peptide stimulated significant release compared with assay buffer alone (P0.0189). As a positive control, eosino- phils were treated with IgG-coupled sepharose beads, and release of over 4 ng/ml cys-LT was detected (data not shown).

Article Snippet: Mouse monoclonal antibodies (mAb) to the N-terminal of PAR1 [ATAP2, mouse immunoglobulin G1 (IgG1)] and PAR2 (SAM11, mouse IgG2a) and goat polyclonal antibodies (pAb) directed against the C terminus of each of the PARs were purchased from Santa Cruz (Autogen Bioclear, Wiltshire, UK).

Techniques: Activation Assay, Positive Control

Journal: Journal of leukocyte biology

Article Title: Expression of and functional responses to protease-activated receptors on human eosinophils.

doi: 10.1189/jlb.0702351

Figure Lengend Snippet: Fig. 7. Generation of ROS in human eosinophils in response to PAR activa- tion. Eosinophils were resuspended in assay buffer containing lucigenin and stimulated with PAR ligands and peptides. ROS generation was measured by chemiluminescence, and 1 M PAF was used as a positive control (P0.0041). Thrombin was inactive, whereas trypsin (P0.0062) or the PAR2 peptide (P0.0107) induced significant ROS generation. The PAR2 control peptide also stimulated some ROS generation, but this was significantly reduced compared with the active peptide (P0.0175).

Article Snippet: Mouse monoclonal antibodies (mAb) to the N-terminal of PAR1 [ATAP2, mouse immunoglobulin G1 (IgG1)] and PAR2 (SAM11, mouse IgG2a) and goat polyclonal antibodies (pAb) directed against the C terminus of each of the PARs were purchased from Santa Cruz (Autogen Bioclear, Wiltshire, UK).

Techniques: Positive Control, Control

Journal: PLoS ONE

Article Title: The Catalytic Subunit of Protein Phosphatase 1 Gamma Regulates Thrombin-Induced Murine Platelet α IIb β 3 Function

doi: 10.1371/journal.pone.0008304

Figure Lengend Snippet: ( A ) Lysate from resting washed platelets was evaluated by SDS-PAGE and immunoblotted with anti-PAR3 antibody (∼43 kDa). The blot was stripped and reprobed with anti- PAR4 (∼38 kDa) and then with anti-actin (loading) antibodies. Blots are representative of two experiments. Washed platelets were incubated with anti-JON/A PE antibody ( B ) or anti-α IIb FITC antibody ( C ) or anti-P-selectin antibody ( D ) and subjected to flow cytometry. Results are expressed as ±SEM of 5–6 experiments. Compared to thrombin stimulated WT platelets, the decreased binding of JON/A antibody to PP1cγ −/− platelets was significant at *p = 0.003.

Article Snippet: Antibodies to theα, and γ isoforms of PP1c, PAR3 and PAR4 were purchased from Santacruz Biotechnology (Santacruz, CA). β isoform of PP1c was from Upstate Biotechnology/Millipore (Billerica, MA).

Techniques: SDS Page, Incubation, Flow Cytometry, Binding Assay