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 (1μM) for 72 hours. 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 hours 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 hours. 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 hours. 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 (1mM) for 48 hours. 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.

Fig 3: PARG is essential for cell survival.a, Diagram of full-length PARG was presented with the indicated gRNAs which target different regions in the C-terminal catalytic domain. The boundary of the catalytic domain was depicted based on Uniprot annotation. b, Immunoblotting was conducted to confirm the loss of PARG in PARG complete/conditional knockout (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 (100nM) 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 hours, 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 hours.

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: 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 of HEK 293 A PARG/PARP1/2 TKO and PARP1 reconstitution cells to PARGi. Cells were treated with different doses of PARGi for 72 hr, and cell viability was determined by the CellTiter-Glo assay. (c) Prolonged PARGi treatment induces pADPr throughout the cell cycle in PARG KO cells. HEK293A WT and PARG KO cells were treated with PARGi (10 µM) for the indicated time and then fixed and stained with anti-pADPr antibody and violet. (d) Immunoblotting of γH2A.X signals and other indicated proteins and modifications induced by prolonged PARGi +/-MMS treatment. Figure 4—source data 1.Original file for the western blot analysis in Figure 4. Figure 4—source data 2.PDF containing Figure 4 and original scans of the relevant western blot analysis with highlighted bands and sample labels.