recombinant his parp1 protein  (MedChemExpress)

Journal: Science Advances

Article Title: EZH2 directly methylates PARP1 and regulates its activity in cancer

doi: 10.1126/sciadv.adl2804

Figure Lengend Snippet: ( A to C ) C4-2 cells (A), DU145 cells (B), or CRPC PDX LuCaP 35CR tissues (C) were lysed and collected for IP with anti-EZH2, PARP1, or immunoglobulin G (IgG), followed by IB analysis with indicated antibodies. ( D ) Purified proteins of GST-tagged EZH2 and His-tagged PARP1 were subjected to GST pull-down, followed by IB analysis. ( E ) Domain architecture of the EZH2 protein and its truncated mutants generated by IBS 2.0 . The homology domain 1 (H1) contains WDB domain, while the homology domain 2 (H2) contains the first SANT domain. ( F ) Co-IP of Flag-tagged PARP1 with full-length or truncated mutants of Myc-tagged EZH2, followed by IB analysis. ( G ) Domain organization of the PARP1 protein and its truncations generated by IBS 2.0 . ND, N-terminal domain; MD, middle domain; CD, C-terminal domain. ( H ) Co-IP of Myc-tagged EZH2 with truncated mutants of GST-tagged PARP1, followed by IB analysis. ( I ) Top, C4-2 cells were infected with control or two independent shEZH2 lentiviruses for 72 hours and subjected to Western blotting (WB). Bottom, quantification of relative PAR abundance normalized by H3 protein intensity. ( J ) Top, C4-2 cells were treated with either dimethyl sulfoxide (DMSO) or indicated doses of EZH2 inhibitor (EZH2i) for 3 days. Co-IP was performed with anti-PARP1 antibody and IB with indicated antibodies. Bottom, quantification of relative PARP1 autoPARylation abundance normalized by PARP1 protein intensity in IP samples. ( K ) Top, C4-2 cells were treated with either DMSO or indicated doses of EZH2i for 3 days. Co-IP was performed with anti-H2B antibody and IB with indicated antibodies. Bottom, quantification of relative H2B PARylation abundance normalized by H2B protein intensity in IP samples. All error bars represent means ± SD from n = 3 biologically independent experiments. P values were determined by unpaired two-tailed t test. ** P < 0.01, *** P < 0.001.

Article Snippet: Following procedures previously described in , 1 μg of recombinant GST-tagged EZH2 protein was mixed with 1 μg of recombinant His-tagged PARP1 protein in 1 ml of NP-40 lysis buffer (Thermo Fisher Scientific) with protease and phosphatase inhibitor cocktails (Thermo Fisher Scientific).

Techniques: Purification, Generated, Co-Immunoprecipitation Assay, Infection, Control, Western Blot, Two Tailed Test

Journal: Science Advances

Article Title: EZH2 directly methylates PARP1 and regulates its activity in cancer

doi: 10.1126/sciadv.adl2804

Figure Lengend Snippet: ( A ) Co-IP with anti-methylated lysine (Methyl K) antibody was performed in C4-2 and DU145 cells, followed by IB with anti-PARP1 or anti-H3 antibodies. Rabbit IgG was used as a negative control. ( B ) HEK293T and C4-2 cells were treated with either DMSO or indicated doses of EPZ-6438 for 3 days before co-IP with anti-PARP1 antibody. Methylation was detected by IB with anti–Methyl K antibody. ( C ) ESI-MS/MS fragmentation spectra of unmodified and methylated K486- and K607-containing peptides from proteolytically digestion by chymotrypsin. ( D ) Relative methylation percentage of the indicated lysine sites in HEK293T cell with or without EZH2i treatment by MS data analysis. ( E ) HEK293T cells were transfected with Flag-tagged PARP1-WT or -K607A. Co-IP was performed with anti-Flag antibody followed by IB with anti–Methyl K antibody, or reversely IP with anti–Methyl K antibody and IB with anti-Flag antibody. ( F ) C4-2 cells stably expressing Flag-tagged PARP1-WT or -K607A were subjected to IP with anti-Flag antibody and followed by IB with anti-K607me1 antibody. ( G ) C4-2 cells were treated with either DMSO or EZH2i EPZ-6438 for 3 days. IP was performed with anti-PARP1 antibody and IB with anti-K607me1 antibody. ( H ) Rescue assay followed by Co-IP to determine PARP1 K607 methylation level in EZH2-deficient C4-2 cells overexpressed with either WT or H689A-mutant EZH2. ( I ) Cell lysates were collected from EZH2 +/+ , EZH2 −/− , EED +/+ , and EED −/− XEN cells and subjected to Co-IP assay followed by WB to detect PARP1 K607me1. ( J ) Co-IP with anti-EZH2 followed by IB with anti-PAR, and Co-IP with anti-PARP1 followed by IB with anti-K607me1 were performed in C4-2 cells treated with 5 nM BMN 673 for 48 hours.

Article Snippet: Following procedures previously described in , 1 μg of recombinant GST-tagged EZH2 protein was mixed with 1 μg of recombinant His-tagged PARP1 protein in 1 ml of NP-40 lysis buffer (Thermo Fisher Scientific) with protease and phosphatase inhibitor cocktails (Thermo Fisher Scientific).

Techniques: Co-Immunoprecipitation Assay, Methylation, Negative Control, Tandem Mass Spectroscopy, Transfection, Stable Transfection, Expressing, Rescue Assay, Mutagenesis

Journal: Science Advances

Article Title: EZH2 directly methylates PARP1 and regulates its activity in cancer

doi: 10.1126/sciadv.adl2804

Figure Lengend Snippet: ( A ) Co-IP followed by IB was performed to measure PARP1 autoPARylation variations in different K607 mutant cell lines. ( B ) In vitro activity assay of recombinant human PARP1-WT and -K607E proteins. Auto-PARylation of PARP1 was examined by SDS–polyacrylamide gel electrophoresis. PARP1-WT or -K607E was incubated without (a) or with 0.1 μM (b), 1 μM (c) dumbbell ssDNA and various concentrations of NAD + . The percentage of PARylated PARP1 was quantified using ImageJ (d). (means ± SD, n = 3). ( C ) Left, representative images of PLA showing XRCC1/PAR foci in the indicated cell lines with or without CPT treatment. Right, quantification of PLA. Stacked bar charts show the percentage of cells with various numbers of XRCC1/PAR foci per nucleus with CPT treatment. Data from five to seven randomly selected fields ( n ≥ 30 cells) in each group were quantified using ImageJ. ( D ) Left, representative images for Comet assay. Right, percentage of tail DNA in total DNA (from 50 cells) was analyzed using the OpenComet software (means ± SD). ( E ) Colony formation assay was performed in the showed cell lines treated with increasing concentrations of CPT (from 0 to 20 nM). Percentages of colonies were assessed after 10 days by counting the number of colonies and normalized to that of untreated cells, which was set at 100% (means ± SD, n = 3). ( F ) Cellular NAD + levels in the indicated C4-2 cells after 30 min treatment with either vehicle (DMSO) or 25 μM CPT. The data shown are reported as percentage of the -EV cell line (means ± SD, n = 3). **** P < 0.0001, *** P < 0.001, ** P < 0.01; n.s., not significant, as determined by two-way analysis of variance (ANOVA) for (B) and (E), or two-tailed t test for (D) and (F).

Article Snippet: Following procedures previously described in , 1 μg of recombinant GST-tagged EZH2 protein was mixed with 1 μg of recombinant His-tagged PARP1 protein in 1 ml of NP-40 lysis buffer (Thermo Fisher Scientific) with protease and phosphatase inhibitor cocktails (Thermo Fisher Scientific).

Techniques: Co-Immunoprecipitation Assay, Mutagenesis, In Vitro, Activity Assay, Recombinant, Polyacrylamide Gel Electrophoresis, Incubation, Single Cell Gel Electrophoresis, Software, Colony Assay, Two Tailed Test

Journal: Science Advances

Article Title: EZH2 directly methylates PARP1 and regulates its activity in cancer

doi: 10.1126/sciadv.adl2804

Figure Lengend Snippet: ( A ) Volcano plot showing DEGs between PARP1 KD C4-2 cells stably expressing PARP1-K607A and -WT. Adjusted P < 0.05 was used as the threshold to judge the statistical significance of the difference in gene expression. Red plots represent up-regulated genes (K607A versus WT); blue plots represent down-regulated genes; gray plots represent genes with no significant difference. The names of representative genes relevant to tumor progression were labeled. ( B ) Expression level of MRGs in a public prostate cancer dataset (GSE21032). Boxplot (left) showing the entire signature of MRGs expression pattern and heatmap (right) displaying individual gene expression. Statistical significance determined by two-tailed Student’s t test. Box plots are median and upper and lower quartiles. Whiskers are min and max. ( C ) Top 10 significantly enriched GSEA MSigDB Hallmarks gene sets are shown with normalized enrichment score (NES) and false discovery rate (FDR). ( D ) Graphical view of the enrichment score for the most enriched gene set E2F targets. ( E ) Indicated cells were lysed and collected for IP with anti-E2F1 antibody, followed by IB analysis with indicated antibodies. ( F ) E2F1 promoter luciferase reporter assay in HEK293T cells with transient overexpression of PARP1-K607A or -WT. pGL-basic as a negative control. ( G ) ChIP-qPCR data showing the enrichment of PARP1 and E2F1 at the cyclin A and RAD51 promoters in PARP1 KD C4-2 cells stably expressing PARP1-WT or -K607A. ( H ) RT-qPCR data showing relative mRNA expression of cyclin A and RAD51 in indicated cell lines. All error bars represent means ± SD from n = 3 [for (G) and (H)] or n = 5 [for (F)] biologically independent experiments. P values were determined by unpaired two-tailed t test; n.s., not significant, * P < 0.05, ** P < 0.01, **** P < 0.0001.

Article Snippet: Following procedures previously described in , 1 μg of recombinant GST-tagged EZH2 protein was mixed with 1 μg of recombinant His-tagged PARP1 protein in 1 ml of NP-40 lysis buffer (Thermo Fisher Scientific) with protease and phosphatase inhibitor cocktails (Thermo Fisher Scientific).

Techniques: Stable Transfection, Expressing, Labeling, Two Tailed Test, Luciferase, Reporter Assay, Over Expression, Negative Control, Quantitative RT-PCR

Journal: Science Advances

Article Title: EZH2 directly methylates PARP1 and regulates its activity in cancer

doi: 10.1126/sciadv.adl2804

Figure Lengend Snippet: ( A ) Proliferation of indicated C4-2 cell lines. ( B ) Wound healing assay was conducted to evaluate the migration potential of PARP1 KD C4-2 cells stably expressing EV, WT, K607A, or K607R. The healing of wounded cell layer was monitored under a microscope every 24 hours. Bar chart showing the rate of filling of the scratched area by cells. ( C ) Boyden chamber invasion assay was performed to determine the invasive capability of PARP1 KD C4-2 cells stably expressing EV, WT, K607A, or K607R. Graph showing the number of invaded cells passing through Matrigel at 24 hours. ( D ) Soft agar colony formation assay was performed to monitor anchorage-independent growth ability of PARP1 KD C4-2 cells stably expressing EV, WT, or K607A. ( E ) Intravascular dissemination of C4-2B GFP-luciferase cells stably expressing EV, WT, or K607A in CAM assay. ( F ) qPCR quantification of human genomic DNA content at the Alu gene locus in chicken livers (left) and lungs (right) from CAM tumor models. All error bars represent means ± SD from n = 3 [for (B) and (C)] or n = 5 [for (A), (D), and (F)] biologically independent experiments. P values were determined by unpaired two-tailed t test [for (B), (C), (D), and (F)] or two-way ANOVA for (A). ns, not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Following procedures previously described in , 1 μg of recombinant GST-tagged EZH2 protein was mixed with 1 μg of recombinant His-tagged PARP1 protein in 1 ml of NP-40 lysis buffer (Thermo Fisher Scientific) with protease and phosphatase inhibitor cocktails (Thermo Fisher Scientific).

Techniques: Wound Healing Assay, Migration, Stable Transfection, Expressing, Microscopy, Invasion Assay, Soft Agar Assay, Luciferase, Chick Chorioallantoic Membrane Assay, Two Tailed Test

Journal: Science Advances

Article Title: EZH2 directly methylates PARP1 and regulates its activity in cancer

doi: 10.1126/sciadv.adl2804

Figure Lengend Snippet: ( A ) Seesaw model for combinational strategy of EZH2 and PARP1 inhibition. ( B and C ) Quantification of synergistic potency and efficiency of EPZ-6438 and BMN 673 in C4-2 (B) and PC3 cells (C) by MuSyc . Left, dose-response surface. Middle, dose-response curve for BMN 673. Right, dose-response curve for EPZ-6438. The parameter alpha quantifies how the effective dose of one drug is altered by the presence of the other. In the case of synergistic potency, alpha >1. The parameter beta is defined as the percent increase in a drug combination’s effect beyond the most efficacious single drug. In the case of synergistic efficacy, beta >0. ( D ) C4-2 cell viability in the presence of the indicated concentrations of PARPi (talazoparib) along with DMSO or EZH2i (EPZ-6438). ( E ) PARP1 KD C4-2 cells with stably expression of PARP1-WT, -K607A, or -K607R were subjected to colony formation assay in the presence of the indicated concentrations of PARPi (talazoparib). ( F to H ) LuCaP 35CR PDX tumor growth in nude severe combined immunodeficiency mice received vehicle, EPZ-6438, BMN 673, or both for 28 days. Tumor weights (F) were measured at the end point. Tumor volumes (G) and body weights (H) were monitored on the indicated days. ( I ) WB for on-target validation. Tumor tissues were lysed and blotted with indicated antibodies. Protein levels were quantified and normalized against H3. ( J ) Left, representative images of IHC staining for Ki-67 and cleaved-caspase 3 (CC3). Right, quantification of IHC images by ImageJ. Error bars, means ± SD, n = 5, **** P < 0.0001, *** P < 0.001, ** P < 0.01, * P < 0.05; ns, not significant, as determined by unpaired two-tailed t test for (F), (I), and (J) or two-way ANOVA for (G).

Article Snippet: Following procedures previously described in , 1 μg of recombinant GST-tagged EZH2 protein was mixed with 1 μg of recombinant His-tagged PARP1 protein in 1 ml of NP-40 lysis buffer (Thermo Fisher Scientific) with protease and phosphatase inhibitor cocktails (Thermo Fisher Scientific).

Techniques: Inhibition, Stable Transfection, Expressing, Colony Assay, Immunohistochemistry, Two Tailed Test

Journal: Science Advances

Article Title: EZH2 directly methylates PARP1 and regulates its activity in cancer

doi: 10.1126/sciadv.adl2804

Figure Lengend Snippet: EZH2 directly interacts with and methylates PARP1 at lysine 607. EZH2 inhibition or PARP1 K607 mutation increases PARP1 poly(ADP-ribose) polymerase activity, fine-tunes PARP1 functions in DDR, transcription, and tumor progression.

Article Snippet: Following procedures previously described in , 1 μg of recombinant GST-tagged EZH2 protein was mixed with 1 μg of recombinant His-tagged PARP1 protein in 1 ml of NP-40 lysis buffer (Thermo Fisher Scientific) with protease and phosphatase inhibitor cocktails (Thermo Fisher Scientific).

Techniques: Inhibition, Mutagenesis, Activity Assay