DePARylation is critical for S phase progression and cell survival
- Department of Experimental Radiation Oncology, The University of Texas MD Anderson Cancer Center, United States
- Institute for Personalized Cancer Therapy, The University of Texas MD Anderson Cancer Center, United States
Figures
Figure 1 with 1 supplement
PARG loss sensitizes cells to PARGi.
(a) Ranking of co-essential genes with PARGi treatment on the basis of a DrugZ analysis of the results of CRISPR/Cas9 screening with a DDR library in HEK293A cells. The NormZ score was used to …
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Figure 1—source data 1
DrugZ analysis results of CRISPR/Cas9 screening with DDR library in PARGi treated HEK293A cells.
- https://cdn.elifesciences.org/articles/89303/elife-89303-fig1-data1-v1.xlsx
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Figure 1—source data 2
Original file for the western blot analysis in Figure 1B and Figure 1—figure supplement 1D.
- https://cdn.elifesciences.org/articles/89303/elife-89303-fig1-data2-v1.zip
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Figure 1—source data 3
PDF containing Figure 1C, Figure 1—figure supplement 1D and original scans of the relevant western blot analysis (anti-actin and anti-PARG) with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/89303/elife-89303-fig1-data3-v1.zip
Figure 1—figure supplement 1
PARG KO cells are sensitive to active PARG inhibitors.
(a) Chemical structures of active and inactive PARG inhibitors (PDD 00031705). (b) Another two PARG inhibitors (PDD00017272 and PDD 00017238) were used to determine cell viability in HEK293A WT and …
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Figure 1—figure supplement 1—source data 1
Original file for the western blot analysis in Figure 1—figure supplement 1E.
- https://cdn.elifesciences.org/articles/89303/elife-89303-fig1-figsupp1-data1-v1.zip
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Figure 1—figure supplement 1—source data 2
PDF containing Figure 1—figure supplement 1E and original scans of the relevant western blot analysis (anti-actin, anti-pADPr and anti-PARG) with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/89303/elife-89303-fig1-figsupp1-data2-v1.zip
Figure 2 with 1 supplement
PARGi treatment induces NAD+- and PARP-dependent cell death in PARG KO cells.
(a) HEK293A WT, PARG KO, PARP1/2 DKO, and PARG/PARP1/2 TKO cells were treated with PARGi (1 µM) for 72 hr. The total cell lysates were immunoblotted with the indicated antibodies. (b) HEK293A WT, …
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Figure 2—source data 1
Original file for the western blot analysis and colony formation assay in Figure 2 and Figure 2—figure supplement 1B.
- https://cdn.elifesciences.org/articles/89303/elife-89303-fig2-data1-v1.zip
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Figure 2—source data 2
PDF containing Figure 2 and Figure 2—figure supplement 1B and original scans of the relevant western blot analysis and colony formation assay with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/89303/elife-89303-fig2-data2-v1.zip
Figure 2—figure supplement 1
PARP-dependent pADPr and reduced NAD + may contribute to cell death induced by PARGi treatment in PARG KO cells.
(a) HEK293A WT and PARG KO cells were treated with DMSO or PARGi (10 µM) for 4 hr or 0.01% MMS for 30 min or the combination of PARGi (10 µM) for 4 hr and an additional 30 min with 0.01% MMS. The …
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Figure 2—figure supplement 1—source data 1
Original file for the western blot in Figure 2—figure supplement 1A.
- https://cdn.elifesciences.org/articles/89303/elife-89303-fig2-figsupp1-data1-v1.zip
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Figure 2—figure supplement 1—source data 2
PDF containing Figure 2—figure supplement 1A and original scans of the relevant western blot analysis with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/89303/elife-89303-fig2-figsupp1-data2-v1.zip
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Figure 2—figure supplement 1—source data 3
Original file for the colony formation assay in Figure 2—figure supplement 1.
- https://cdn.elifesciences.org/articles/89303/elife-89303-fig2-figsupp1-data3-v1.zip
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Figure 2—figure supplement 1—source data 4
PDF containing Figure 2—figure supplement 1 and original scans of the relevant colony formation assay with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/89303/elife-89303-fig2-figsupp1-data4-v1.zip
Figure 3 with 1 supplement
PARGi treatment induces S-phase-specific pADPr signaling in PARG KO cells.
(a) HEK293A WT and PARG KO cells were treated with DMSO or 10 µM PARGi for 4 hr or 0.01% MMS for 30 min and then fixed and stained with anti-pADPr antibody or anti-γH2A.X antibody and PI. (b) …
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Figure 3—source data 1
Original file for the colony formation assay in Figure 3.
- https://cdn.elifesciences.org/articles/89303/elife-89303-fig3-data1-v1.zip
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Figure 3—source data 2
PDF containing Figure 3 and original scans of the relevant colony formation assay with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/89303/elife-89303-fig3-data2-v1.zip
Figure 3—figure supplement 1
Treatment with DNA damaging agents did not induce S-phase-specific pADPr signaling.
(a) HEK293A WT and PARG KO cells were treated with DMSO, 10 µM PARGi for 4 hr, or other DNA damaging agents and then fixed and stained with PI and anti-pADPr antibody (upper panel) or anti-γH2A.X …
Figure 4 with 1 supplement
Prolonged PARGi treatment induces pADPr throughout the cell cycle and DDR in PARG KO cells.
(a) Immunoblots of chromatin-bound PARP1 and PARylated proteins in HEK293A WT and PARG KO cells treated with PARGi (10 µM) for 4 hr. PARylated proteins were enriched by Af1521 beads. (b) Sensitivity …
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Figure 4—source data 1
Original file for the western blot analysis in Figure 4.
- https://cdn.elifesciences.org/articles/89303/elife-89303-fig4-data1-v1.zip
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Figure 4—source data 2
PDF containing Figure 4 and original scans of the relevant western blot analysis with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/89303/elife-89303-fig4-data2-v1.zip
Figure 4—figure supplement 1
Uncontrolled S phase pADPr accumulation eventually leads to DNA damage and cell death.
(a) Immunoblots of soluble and chromatin-bound PARP1/2 levels in different knockout cells treated with MMS, PARPi, or both. (b) PARG/PARP1/2 TKO cells, reconstituted with PARP1 WT or …
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Figure 4—figure supplement 1—source data 1
Original file for the western blot analysis in Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/89303/elife-89303-fig4-figsupp1-data1-v1.zip
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Figure 4—figure supplement 1—source data 2
PDF containing Figure 4—figure supplement 1 and original scans of the relevant western blot analysis with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/89303/elife-89303-fig4-figsupp1-data2-v1.zip
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Figure 4—figure supplement 1—source data 3
Original images for alkaline comet assay in Figure 4—figure supplement 1.
- https://cdn.elifesciences.org/articles/89303/elife-89303-fig4-figsupp1-data3-v1.zip
Figure 5 with 1 supplement
CRISPR screening identifies regulators of pADPr and cell viability.
(a) Workflow of whole-genome CRISPR screens. For FACS-based CRISPR screening, 5 days after puromycin selection, cells were treated with PARGi (10 µM) for 4 hr and then stained with anti-pADPr …
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Figure 5—source data 1
NormZ score of FACS-based TKOv3 library screen conducted with PARG KO cells with or without PARGi.
- https://cdn.elifesciences.org/articles/89303/elife-89303-fig5-data1-v1.xlsx
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Figure 5—source data 2
NormZ score of cell viability–based TKOv3 library screen conducted with HEK293A cells or PARG KO cells treated with PARGi.
- https://cdn.elifesciences.org/articles/89303/elife-89303-fig5-data2-v1.xlsx
Figure 5—figure supplement 1
Proteins involved in pADPr regulation contribute to PARGi sensitivity.
(a) NormZ scores of sgRNA abundance between top and bottom groups in FACS-based anti-pADPr screens with HEK293A treated with PARGi (10 µM) for 4 hr. (b) ARH3 loss and (c) HPF1 loss show modestly …
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Figure 5—figure supplement 1—source data 1
NormZ score of FACS-based TKOv3 library screen conducted with HEK293A cells treated with PARGi.
- https://cdn.elifesciences.org/articles/89303/elife-89303-fig5-figsupp1-data1-v1.xlsx
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Figure 5—figure supplement 1—source data 2
Original file for the western blot analysis in Figure 5—figure supplement 1.
- https://cdn.elifesciences.org/articles/89303/elife-89303-fig5-figsupp1-data2-v1.zip
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Figure 5—figure supplement 1—source data 3
PDF containing Figure 5—figure supplement 1 and original scans of the relevant western blot analysis with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/89303/elife-89303-fig5-figsupp1-data3-v1.zip
Figure 6 with 2 supplements
PARG expression is a potential marker for PARGi sensitivity.
(a) PARG mRNA level comparison between sensitive (RMGI, KURAMOCHI, and OVMANA) and resistant cells (COV362, COV318, OV56, OVISE, OVSAHO, CAOV3, and OVCAR3) from Pillay’s work on the basis of CCLE …
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Figure 6—source data 1
Original file for the western blot analysis and colony formation assay in Figure 6.
- https://cdn.elifesciences.org/articles/89303/elife-89303-fig6-data1-v1.zip
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Figure 6—source data 2
PDF containing Figure 6 and original scans of the relevant western blot analysis and colony formation assay with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/89303/elife-89303-fig6-data2-v1.zip
Figure 6—figure supplement 1
HR deficiency renders cells sensitive to PARGi.
(a) Results of clonogenic assays conducted with RPE1 FLAG-Cas9 P53/BRCA1 DKO and PRE FLAG-Cas9 P53 KO cells with the indicated treatment for 14 days. (b) Results of clonogenic assays conducted with …
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Figure 6—figure supplement 1—source data 1
Original file for the western blot analysis and colony formation assay in Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/89303/elife-89303-fig6-figsupp1-data1-v1.zip
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Figure 6—figure supplement 1—source data 2
PDF containing Figure 6—figure supplement 1 and original scans of the relevant western blot analysis and colony formation assay with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/89303/elife-89303-fig6-figsupp1-data2-v1.zip
Figure 6—figure supplement 2
PARG loss is a robust marker of PARGi sensitivity.
(a) Immunoblot of PARG in cells treated with PARG shRNA to knock down PARG. (b) Results of clonogenic assays conducted in PARG knockdown cells. PARG was knocked down in the inducible BRCA1 depletion …
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Figure 6—figure supplement 2—source data 1
Original file for the western blot analysis and colony formation assay in Figure 6—figure supplement 2.
- https://cdn.elifesciences.org/articles/89303/elife-89303-fig6-figsupp2-data1-v1.zip
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Figure 6—figure supplement 2—source data 2
PDF containing Figure 6—figure supplement 2 and original scans of the relevant western blot analysis and colony formation assay with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/89303/elife-89303-fig6-figsupp2-data2-v1.zip
Figure 7 with 2 supplements
PARG is essential for cell survival.
(a) Diagram of full-length PARG was presented with the indicated gRNAs (gRNA#3 and 4) which target different regions in the C-terminal catalytic domain. The boundary of the catalytic domain was …
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Figure 7—source data 1
Original file for the western blot analysis and colony formation assay in Figure 7.
- https://cdn.elifesciences.org/articles/89303/elife-89303-fig7-data1-v1.zip
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Figure 7—source data 2
PDF containing Figure 7 and original scans of the relevant western blot analysis and colony formation assay with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/89303/elife-89303-fig7-data2-v1.zip
Figure 7—figure supplement 1
KO cells were validated by DNA sequencing.
gDNA sequences were provided for each KO clones. The WT reference sequence was also provided.
Figure 7—figure supplement 2
DePARylation activity of PARG is essential for cell survival.
(a) Immunoblotting of PARG using two independent antibodies that recognize the C-terminus of PARG in PARG KO cells. The immunogens and the corresponding amino acid regions were shown (left). (b) …
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Figure 7—figure supplement 2—source data 1
Original file for the western blot in Figure 7—figure supplement 2A.
- https://cdn.elifesciences.org/articles/89303/elife-89303-fig7-figsupp2-data1-v1.zip
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Figure 7—figure supplement 2—source data 2
PDF containing Figure 7—figure supplement 2A and original scans of the relevant western blot analysis with highlighted bands and sample labels.
- https://cdn.elifesciences.org/articles/89303/elife-89303-fig7-figsupp2-data2-v1.zip
Additional files
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Supplementary file 1
The CRISPR-Cas9 screening results.
(a) NormZ score of DDR library screen conducted with HEK293A cells treated with PARGi. (b) NormZ score of FACS-based TKOv3 library screen conducted with HEK293A cells treated with PARGi. (c) NormZ score of FACS-based TKOv3 library screen conducted with PARG KO cells. (d) NormZ score of FACS-based TKOv3 library screen conducted with PARG KO cells treated with PARGi. (e) NormZ score of cell viability–based TKOv3 library screen conducted with HEK293A cells treated with PARGi. (f) NormZ score of cell viability–based TKOv3 library screen conducted with PARG KO cells treated with PARGi.
- https://cdn.elifesciences.org/articles/89303/elife-89303-supp1-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/89303/elife-89303-mdarchecklist1-v1.docx
