Fig 2: 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, PARG KO, PARP1/2 DKO, and PARG/PARP1/2 TKO cells were treated with DMSO or 10 µM PARGi for 4 hr and then fixed and stained with anti-pADPr antibody and propidium iodide (PI). (c) HEK293A WT, PARG KO, PARP1/2 DKO clls, and PARG/PARP1/2 TKO cells were treated with different doses of PARGi for 72 hr. Cell viability was determined by the CellTiter-Glo assay. (d) Relative NAD +level in HEK293A WT and PARG KO cells with the indicated treatment for 48 hr. PARGi, 10 µM; PARPi, 10 µM; NAM, 100 µM; NMN, 1 mM; FK866, 10 nM. (e) PARG KO cells were treated with PARGi (10 µM) or PARGi and NAM (100 µM) or NMN (1 mM) for 48 hr. The total cell lysates were immunoblotted with the indicated antibodies. (f) Results of clonogenic assays conducted using HEK293A PARG KO cells treated with PARGi (500 nM) or PARGi and NAM (100 µM) or NMN (1 mM) for 7 days. 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. 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.

Fig 3: 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 data. (b) Clonogenic assay results of control and PARG knockdown HeLa cells with PARGi (2 µM) treatment for 7 days. shRNA knockdown efficiency was confirmed by an immunoblot of PARG. (c) Ranked PARG expression level in ovarian cancer cell lines based on the CCLE database. The sensitive and resistant cells from Pillay’s work were labeled. (d) Clonogenic assay results of OVCAR3 and RMUGS treated with or without PARGi (2 µM). (e) Clonogenic assay results of RMUGS treated with PARGi (2 µM), PARPi (2 µM), or both. (f) Left: Representative images of PARG IHC staining in breast and ovarian tissues and tumor samples to determine the PARG expression level. The summary is listed at the bottom. Scale bar, 200 µm. Right: The scatter plot of mean immunostaining intensity of PARG in each sample. The mean of each group was plotted. Figure 6—source data 1.Original file for the western blot analysis and colony formation assay in Figure 6. 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.

Fig 4: 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 depicted based on Uniprot annotation. gRNA#1 and gRNA#2 were used previously to generate the aforementioned HEK293A- and HeLa-derived PARG KO cells, respectively; while gRNA#3 and gRNA#4 were used to generate PARG complete/conditional knockout (cKO) in the presence of olaparib in HEK293A and HeLa cells. (b) Immunoblotting was conducted to confirm the loss of PARG in PARG cKO cells derived from HEK293A and HeLa cells, which were cultured in the presence of 100 nM olaparib. (c) Clonogenic assay results of WT and PARG cKO cells treated with or without PARPi (100 nM) for 7 days. (d) Left: The immunoblots to confirm reconstitution with WT PARG or catalytic inactivation PARG in HEK293A PARG cKO cells. Right: Results of clonogenic survival assay with HEK293A PARG cKO cells reconstituted with WT or catalytic inactivation mutant of PARG for 7 days. (e) Representative clonogenic results conducted in HEK293A PARG cKO cells treated with NAM (100 µM) or NMN (1 mM) for 7 days. (f) HEK293A PARG cKO cells were synchronized with double thymidine block (DTB). Cells remained with DTB or were released from DTB for 4 hr, and then fixed and stained with anti-pADPr antibody and FxCycle Violet dye. (g) Immunoblots of soluble and chromatin-bound PARP1 and pADPr levels in HEK293A WT and PARG cKO cells treated with DMSO or olaparib (10 µM) for 2 hr. Figure 7—source data 1.Original file for the western blot analysis and colony formation assay in Figure 7. 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.

Fig 5: 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 determine a possible synthetic lethality gene under PARGi treatment. Drug-sensitive genes were marked in red; drug-resistant genes were marked in blue on the basis of the false discovery rate (FDR, 0.05 cut-off). (b) HEK293A WT cells and HEK293A PARG KO cells were treated with different doses of PARGi for 72 hr. Cell viability was determined by the CellTiter-Glo assay. (c) HEK293A PARG KO cells, re-constituted with either full-length PARG or catalytic domain mutation of PARG, were treated with different doses of PARGi for 72 hr. Cell viability was determined by the CellTiter-Glo assay. Figure 1—source data 1.DrugZ analysis results of CRISPR/Cas9 screening with DDR library in PARGi treated HEK293A cells. Figure 1—source data 2.Original file for the western blot analysis in Figure 1B and Figure 1—figure supplement 1D. 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.