Development and characterization of new tools for detecting poly(ADP-ribose) in vitro and in vivo
- Laboratory of Signaling and Gene Regulation, Cecil H. and Ida Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center, United States
- Division of Basic Research, Department of Obstetrics and Gynecology, University of Texas Southwestern Medical Center, United States
- Program in Genetics, Development, and Disease, Graduate School of Biomedical Sciences, University of Texas Southwestern Medical Center, United States
Figures
Figure 1 with 1 supplement
Development of ddGFP-based fluorescent sensors to measure PAR levels.
(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 …
Figure 1—figure supplement 1
Characterization of fluorescence-based PAR-Trackers using biochemical assays.
(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 …
Figure 2 with 2 supplements
Tracking PAR accumulation in live cells using PAR-T ddGFP.
(A, B) Live-cell imaging assay to track PAR formation in response to H2O2 in 293T cells subjected to Dox-induced PAR-T ddGFP expression. The cells were treated with 20 µM PJ34 for 2 hr prior to …
Figure 2—figure supplement 1
Characterization of fluorescence-based PAR-Trackers using live-cell imaging.
(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 …
Figure 2—video 1
Tracking the levels of PAR in cells.
Live-cell tracking of PAR formation in response to H2O2. 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 …
Figure 3
Tracking the spatial accumulation of PAR using PAR-T ddGFP.
(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 …
Figure 4 with 2 supplements
Tracking PAR accumulation in response to PARP or PARG inhibition, or PARP1 or PARP2 knockdown using PAR-T NanoLuc.
(A) Schematic diagram of the plasmid constructs used to express the split Nano luciferase PAR-Tracker (PAR-T NanoLuc) in mammalian cells. The constructs contain DNA segments encoding (1) Flag tag …
Figure 4—figure supplement 1
Characterization of a luminescence-based PAR-Tracker.
(A) Quantitative bioluminescence imaging of cell extracts isolated from HEK 293T cells subjected to expression of the indicated PAR binding domains conjugated to split firefly luciferase fragments. …
Figure 4—figure supplement 2
Measuring the levels of PAR in a limited number of cells using PAR-T NanoLuc.
(A and B) Bioluminescence imaging (A) of the indicated number of 231-PAR-T NanoLuc cells. Each bar in the graph in (B) represents the mean ± SEM of the relative levels of the ratio of luminescence …
Figure 5
Tracking PAR accumulation in response to UV-induced DNA damage using PAR-T NanoLuc.
(A) Western blot analysis of 231-PAR-T NanoLuc cells treated with Niraparib or PARG inhibitor prior to UV radiation. (B, C) Bioluminescence imaging (B) of 231-PAR-T NanoLuc cells treated with 20 µM …
Figure 6
Comparison of assay performance using the PAR-T sensor and conventional PAR detection reagents.
(A) Quantitative analysis of Western blot analysis and fluorescence measurements (shown in Figure 1G) of the time course of in vitro PAR degradation using recombinant ARH3. Each line plot in the …
Figure 7
Tracking the levels of PAR during adipogenesis.
(A, B) Bioluminescence imaging of PAR-T NanoLuc (A) and unsplit NanoLuc (B) in 3T3-L1 cells subjected to adipogenic differentiation for 12 or 24 hr. (C, D) Quantification of signals from PAR-T …
Figure 8 with 1 supplement
Measuring the levels of PAR in tissues in living animals using PAR-T NanoLuc.
(A) Schematic diagram of the in vivo studies performed using 231-PAR-T NanoLuc cells. (B, C) Bioluminescence imaging (B) of xenograft tumors formed using 231-PAR-T NanoLuc cells. The tumors were …
Figure 8—figure supplement 1
Tracking the levels of PAR in tissues in living animals using PAR-T NanoLuc.
(A) Bioluminescence imaging of firefly luciferase of tumors formed using 231-PAR-T NanoLuc cells as shown in Figure 8B. (B–D) Time course of Bioluminescence imaging of 231-PAR-T NanoLuc cells …
Tables
Table 1
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. …
| Nomenclature used in the manuscript | ARBDs used with fluorescent or luminescent protein fragments | Activity in vitro | Activity in cells | Figures | |
|---|---|---|---|---|---|
| Fluorescent sensors | ddGFP-A | ddGFP-B | |||
| PAR-T ddGFP | WWE from RNF146 | WWE from RNF146 | +++ | ++++ | Figure 1—figure supplement 1D, E |
| Macrodomain from AF1521 | Macrodomain from AF1521 | ++ | N.D. | Figure 1—figure supplement 1D, E | |
| PBZ from APLF | PBZ from APLF | + | N.D. | Figure 1—figure supplement 1D, E | |
| Macrodomain from MH2A.1 | Macrodomain from MH2A.1 | + | N.D. | Figure 1—figure supplement 1D, E | |
| WWE from RNF146 | Macrodomain from AF1521 | +++ | N.D. | Figure 1—figure supplement 1D, E | |
| Macrodomain from AF1521 | WWE from RNF146 | ++++ | – | Figure 1—figure supplement 1D, E | |
| Firefly luciferase-based sensors | N-terminal of Firefly luciferase (FLucN) | C-terminal of Firefly luciferase (FLucC) | |||
| PAR-T fLuc | WWE from RNF146 | WWE from RNF146 | N.D. | + | Figure 4—figure supplement 1C |
| Macrodomain from AF1521 | Macrodomain from AF1521 | N.D. | – | Figure 4—figure supplement 1C | |
| WWE from RNF146 | Macrodomain from AF1521 | N.D. | – | Figure 4—figure supplement 1C | |
| Macrodomain from AF1521 | WWE from RNF146 | N.D. | – | Figure 4—figure supplement 1C | |
| Nano luciferase-based sensors | N-terminal of Nano luciferase (NanoLucN) | C-terminal of Nano luciferase plus LssmOrange (NanoLucC) | |||
| PAR-T NanoLuc | WWE from RNF146 | WWE from RNF146 | N.D. | ++++ | Figure 4C |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Escherichia coli) | BL21(DE3-pLysis) | Thermo Fisher Scientific | Cat. no. C606010 | |
| Cell line (Homo sapiens) | MCF-7 (female adult breast cancer) | ATCC | RRID:CVCL_0031 | |
| Cell line (H. sapiens) | 3T3-L1 (male adult preadipocyte) | ATCC | RRID:CVCL_0123 | |
| Cell line (H. sapiens) | HeLa (female adult cervical cancer) | ATCC | RRID:CVCL_0030 | |
| Cell line (H. sapiens) | MDA-MB-231 Luc (female adult breast cancer) | Obtained from Dr. Srinivas Malladi, UT Southwestern | ||
| Cell line (H. sapiens) | HEK 293T (normal embryonic kidney) | ATCC | RRID:CVCL_0063 | |
| Transfected construct (H. sapiens) | PARP1 siRNA | Sigma-Aldrich | Cat. no. SASI_Hs01_0033277 | |
| Transfected construct (H. sapiens) | PARP2 siRNA | Sigma-Aldrich | Cat. no. SASI_Hs01_0013-1488 | |
| Antibody | Anti-poly-ADP-ribose binding reagent (rabbit monoclonal; IgG Fc) | MilliporeGibson et al., 2017 | Cat. no. MABE1013 | WB (5 µg/ml)IF (1:500) |
| Antibody | PARP-1 (rabbit polyclonal) | Active Motif | Cat. no. 39559 | WB (1:1000) |
| Antibody | β-tubulin (rabbit polyclonal) | Abcam | Cat. no. ab6046RRID: AB_2210370 | WB (1:1000) |
| Antibody | HRP-conjugated anti-rabbit IgG (goat polyclonal) | Pierce | Cat. no. 31460RRID: AB_228341 | WB (1:5000) |
| Antibody | Alexa Fluor 594-conjugated anti-rabbit IgG (donkey polyclonal) | Thermo Fisher Scientific | Cat. no. A-21207RRID: AB_141637 | IF (1:500) |
| Commercial assay or kit | Nano-Glo Live Cell Assay System | Promega | Cat. no. N2011 | |
| Commercial assay or kit | Nano-Glo Luciferase Assay System | Promega | Cat. no. N1110 | |
| Commercial assay or kit | Luciferase Assay System | Promega | Cat. no. E1500 | |
| Commercial assay or kit | PAR ELISA Kit | Cell Biolabs | Cat. no. XDN-5114 | |
| Chemical compound, drug | PJ34 | Enzo | Cat. no. ALX-270 | In cells (20 µM) |
| Chemical compound, drug | Niraparib | MedChem Express | Cat. no. HY-10619 | In cells (20 µM)In vivo (25 mg/kg body weight) |
| Chemical compound, drug | PDD00017273 | MedChem Express | Cat. no. HY-108360 | In cells (20 µM)In vivo (25 mg/kg body weight) |
