ptom20 ddgfp b  (Addgene inc)

Journal: eLife

Article Title: Development and characterization of new tools for detecting poly(ADP-ribose) in vitro and in vivo

doi: 10.7554/eLife.72464

Figure Lengend Snippet: Live-cell tracking of PAR formation in response to H 2 O 2 . Time course images of the live-cell assay shown in Figure 2C . HeLa cells subjected to PAR-T ddGFP expression were treated with 20 µM PJ34 for 2 hr prior to 15 min of treatment with 1 mM H 2 O 2 and live-cell imaging. The 15-min time course of H 2 O 2 treatment was condensed to the time frame shown in the video (1-min sampling interval, 15 frames total). The scale bar is 5 µm.

Article Snippet: The plasmids for Dox-inducible expression of the ddGFP PAR-T constructs were generated using a cDNA for ddGFP-A (Addgene, 40286) or ddGFP-B (Addgene, 40287). cDNAs for the PAR binding domains were amplified from previously published pET19b constructs ( ).

Techniques:

Journal: eLife

Article Title: Development and characterization of new tools for detecting poly(ADP-ribose) in vitro and in vivo

doi: 10.7554/eLife.72464

Figure Lengend Snippet: Live-cell tracking of PAR formation in response to H 2 O 2 . Time course images of the live-cell assay shown in Figure 2C . HeLa cells subjected to PAR-T ddGFP expression were treated with 20 µM PJ34 for 2 hr prior to 15 min of treatment with 1 mM H 2 O 2 and live-cell imaging. The 15-min time course of H 2 O 2 treatment was condensed to the time frame shown in the video (1-min sampling interval, 15 frames total). The scale bar is 5 µm.

Article Snippet: Plate-based fluorescent assays using PAR-T ddGFP were performed in a manner similar to the ELISA assays according to the manufacturer’s protocol (Cell Biolabs, XDN-5114).

Techniques:

Journal: eLife

Article Title: Development and characterization of new tools for detecting poly(ADP-ribose) in vitro and in vivo

doi: 10.7554/eLife.72464

Figure Lengend Snippet: ( A ) SDS-PAGE with Coomassie brilliant blue staining of recombinant ddGFP PAR-Trackers with the indicated ADPR binding domains. ( B ) SDS-PAGE with Coomassie brilliant blue staining of recombinant Flag-tagged PARP-1 and PARP-3 proteins. ( C ) In vitro auto(ADP-ribosyl)ation reactions using recombinant PARP-1 and PARP-3 proteins from ( B ). ( D, E ) Fluorescence measurements ( D ) and heatmap ( E ) of in vitro PARylation detection assays performed using the indicated PAR-binding domains. ( F, G ) Western blot analysis ( F ) and fluorescence measurements ( G ) of in vitro PAR formation using recombinant PARP-1 and the indicated concentrations of NAD + . Each line in the graph in ( G ) represents the mean ± SEM of the relative fluorescence intensity (n=3). ( H ) Fluorescence measurements of in vitro PAR degradation using the indicated concentrations of recombinant ARH3. Each line plot in the graph represents the mean ± SEM of the relative fluorescence intensity (n=3). ( I, J ) Western blot analysis ( I ) and fluorescence measurements ( J ) of PAR in HeLa cell extracts using recombinant ddGFP proteins. The cells were treated with 20 µM PJ34 or 20 µM PARGi for 2 hr before lysis. Each bar in the graph in ( J ) represents the mean ± SEM of the relative fluorescence intensity (n=3, two-way ANOVA, *p<0.05).

Article Snippet: Plate-based fluorescent assays using PAR-T ddGFP were performed in a manner similar to the ELISA assays according to the manufacturer’s protocol (Cell Biolabs, XDN-5114).

Techniques: SDS Page, Staining, Recombinant, Binding Assay, In Vitro, Fluorescence, Western Blot, Lysis

Journal: eLife

Article Title: Development and characterization of new tools for detecting poly(ADP-ribose) in vitro and in vivo

doi: 10.7554/eLife.72464

Figure Lengend Snippet: Nomenclature, composition, and activity of the various PAR-T sensors used in this study. A summary of the PAR-Tracker sensors generated in this study. The activity of these sensors in vitro and in cells, and the corresponding figures in which the activities are described, are indicated. The activities are described as low (+), medium (++), and high (+++, ++++). N.D. not determined.

Article Snippet: Plate-based fluorescent assays using PAR-T ddGFP were performed in a manner similar to the ELISA assays according to the manufacturer’s protocol (Cell Biolabs, XDN-5114).

Techniques: Activity Assay, Generated, In Vitro, Luciferase

Journal: eLife

Article Title: Development and characterization of new tools for detecting poly(ADP-ribose) in vitro and in vivo

doi: 10.7554/eLife.72464

Figure Lengend Snippet: ( A ) Schematic diagram of the fluorescent PAR Trackers (PAR-Ts). ( B ) Schematic diagram of the plasmid constructs used to express the ddGFP PAR-T in bacteria. Chemical structures of a PARylated amino acid, a MARylated amino acid, and the chemical moieties in ADPR that are recognized by the ADPR binding domains. The constructs contain DNA segments encoding (1) His tag (red), (2) ADP-ribose binding domain (yellow), (3) a flexible linker (purple), and (4) ddGFP proteins (white). ( C ) Verification of substrate specificity. Fluorescence measurements of in vitro ADPRylation assays containing the indicated substrates. Each bar in the graph represents the mean ± SEM of the relative fluorescence intensity (n=3, two-way ANOVA, *p<0.0001). ( D, E ) Western blot analysis ( D ) and fluorescence measurements ( E ) of the time course of in vitro PAR formation using recombinant PARP-1. Each line plot in the graph in ( E ) represents mean ± SEM of relative fluorescence intensity (n=3). ( F, G ) Western blot analysis ( F ) and fluorescence measurements ( G ) of the time course of in vitro PAR degradation using recombinant ARH3. Each line plot in the graph in ( G ) represents the mean ± SEM of relative relative fluorescence intensity (n=3).

Article Snippet: Plate-based fluorescent assays using PAR-T ddGFP were performed in a manner similar to the ELISA assays according to the manufacturer’s protocol (Cell Biolabs, XDN-5114).

Techniques: Plasmid Preparation, Construct, Bacteria, Binding Assay, Fluorescence, In Vitro, Western Blot, Recombinant

Journal: eLife

Article Title: Development and characterization of new tools for detecting poly(ADP-ribose) in vitro and in vivo

doi: 10.7554/eLife.72464

Figure Lengend Snippet: ( A ) Schematic diagram of plasmid constructs used to express PAR-T ddGFP in mammalian cells. The constructs contain DNA segments encoding (1) Flag (purple) or HA (dark blue) tags to allow detection of the ddGFPA or ddGFPB protein fragments, respectively, (2) ddGFP-conjugated ADP-ribose binding domains, WWE (yellow) or macrodomain (light blue), (3) IRES (white), (4) mCherry (red), and (5) nuclear localization signal (green). ( B, C ) Live-cell imaging assay to track PAR formation in response to H 2 O 2 . 293T cells subjected to Dox-induced expression of the sensors were treated with 20 µM PJ34 for 2 hr prior to H 2 O 2 treatment. ( B ) Detection by microscopy. The scale bar is 10 µm. ( C ) Each bar in the graph represents the mean ± SEM of the relative levels of the fluorescence intensities (one-way ANOVA; ns=not significant).

Article Snippet: Plate-based fluorescent assays using PAR-T ddGFP were performed in a manner similar to the ELISA assays according to the manufacturer’s protocol (Cell Biolabs, XDN-5114).

Techniques: Plasmid Preparation, Construct, Binding Assay, Live Cell Imaging, Expressing, Microscopy, Fluorescence

Journal: eLife

Article Title: Development and characterization of new tools for detecting poly(ADP-ribose) in vitro and in vivo

doi: 10.7554/eLife.72464

Figure Lengend Snippet: ( A, B ) Live-cell imaging assay to track PAR formation in response to H 2 O 2 in 293T cells subjected to Dox-induced PAR-T ddGFP expression. The cells were treated with 20 µM PJ34 for 2 hr prior to treatment with 1 mM H 2 O 2 for 15 min. The scale bar is 10 µm. Each bar in the graph in ( B ) represents the mean ± SEM of the relative levels of the fluorescence intensity (n=3, one-way ANOVA, *p<0. 01; ns=not significant). ( C, D ) Live-cell tracking of PAR formation in response to H 2 O 2 . HeLa cells subjected to PAR-T ddGFP expression were treated with 20 µM PJ34 for 2 hr prior to treatment with 1 mM H 2 O 2 and live-cell imaging. The scale bar is 10 µm. Each bar in the graph in ( D ) represents the mean ± SD of the relative levels of the fluorescence intensity (n=20 for control and n=21 for PJ34).

Article Snippet: Plate-based fluorescent assays using PAR-T ddGFP were performed in a manner similar to the ELISA assays according to the manufacturer’s protocol (Cell Biolabs, XDN-5114).

Techniques: Live Cell Imaging, Expressing, Fluorescence, Cell Tracking Assay, Control

Journal: eLife

Article Title: Development and characterization of new tools for detecting poly(ADP-ribose) in vitro and in vivo

doi: 10.7554/eLife.72464

Figure Lengend Snippet: ( A, B ) Representative images and quantitative analysis of Z-projections of cancer spheroids formed using MCF-7 cells subjected to Dox-induced expression of PAR-T ddGFP. The spheroids were treated with 20 µM Niraparib for 24 hr prior to imaging. The scale bar is 50 µm. Each bar in the graph in ( B ) represents the mean ± SEM of the relative levels of the fluorescence intensity (n=3 biological replicates containing a total of 8 spheroids for the ddGFPAB control and at least 19 spheroids for PAR-T ddGFP, one-way ANOVA, *p<0.05). ( C, D ) Representative images ( C ) of Z-projections of cancer spheroids formed using MCF-7 cells subjected to Dox-induced expression of the PAR-T ddGFP. The spheroids were treated with 20 µM Niraparib and live-cell imaging was performed at the indicated times. ( Left ) The spheroids were divided into ‘outer’ and ‘core’ sections for quantification as indicated by the white circles. ( Right ) Enlargement of the indicated areas from the left panels (yellow, core; pink, outer) as indicated. Each point in the graph in ( D ) represents the mean ± SEM of the relative levels of PAR-T ddGFP fluorescence intensity normalized to mCherry (n=5, one-way ANOVA, *p<0.05 and **p<0.01).

Article Snippet: Plate-based fluorescent assays using PAR-T ddGFP were performed in a manner similar to the ELISA assays according to the manufacturer’s protocol (Cell Biolabs, XDN-5114).

Techniques: Expressing, Imaging, Fluorescence, Control, Live Cell Imaging

Journal: eLife

Article Title: Development and characterization of new tools for detecting poly(ADP-ribose) in vitro and in vivo

doi: 10.7554/eLife.72464

Figure Lengend Snippet: ( A ) Quantitative analysis of Western blot analysis and fluorescence measurements (shown in ) of the time course of in vitro PAR degradation using recombinant ARH3. Each line plot in the graph represents mean ± SEM of relative intensities (n=3). ( B ) Quantitative analysis of Western blot analysis and bioluminescence imaging (shown in ) of 231-PAR-T NanoLuc cells treated with 20 µM Niraparib or 20 µM PARG inhibitor for 2 hr prior to UV radiation. Each bar in the graph represents the mean ± SEM of the relative intensities (n=3, one-way ANOVA, *p<0.05, **p<0.001, and ***p<0.0001; ns=not significant). ( C ) Measurements of ELISA and fluorescence intensities using 0, 0.625, 1.25, and 2.5 nM concentrations of purified PAR. Each bar in the graph in represents the mean ± SEM of the relative intensities (n=3, paired t-test, *p<0.05, **p<0.01, and ***p<0.001; ns=not significant). ( D ) Immunofluorescence assay using WWE-Fc to measure PAR formation in response to H 2 O 2 using 293T cells. The cells were treated with 20 µM PJ34 (vs. untreated control, ‘Un’) for 2 hr prior to 15 min of treatment with 1 mM H 2 O 2 . The images were collected using a confocal microscope. ( E ) Quantification of the results in ( D ). Each bar in the graph represents the mean ± SEM of the relative levels of the fluorescence intensity of PAR normalized to DAPI (n=3 biological replicates with at least 150 cells in total, one-way ANOVA, ***p<0.0001). ( F ) Representation of the dynamic ranges of PAR-T sensors in comparison to other available PAR detection tools as indicated: (a) Western blotting with WWE-Fc versus live-cell luciferase assay using PAR-T NanoLuc was performed using UV-induced DNA damage in MDA-MB-231 Luc cells (from ( B )); (b) Immunofluorescence with WWE-Fc versus live-cell imaging using PAR-T ddGFP was performed using H 2 O 2 -mediated PARP-1 activation in 293T cells (from ( D )); (c) Western blotting with WWE-Fc versus fluorescence assay with PAR-T ddGFP was performed using ARH3 mediated degradation of PAR in vitro (from ( A )); (d) ELISA versus fluorescence assay with PAR-T ddGFP was performed using immobilized PAR (from ( C )).

Article Snippet: Plate-based fluorescent assays using PAR-T ddGFP were performed in a manner similar to the ELISA assays according to the manufacturer’s protocol (Cell Biolabs, XDN-5114).

Techniques: Western Blot, Fluorescence, In Vitro, Recombinant, Imaging, Enzyme-linked Immunosorbent Assay, Purification, Immunofluorescence, Control, Microscopy, Comparison, Luciferase, Live Cell Imaging, Activation Assay