Journal: eLife

Article Title: DePARylation is critical for S phase progression and cell survival

doi: 10.7554/eLife.89303

Figure Lengend Snippet: ( 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 PARG KO cells. ( c ) HEK293A WT cells and PARG KO cells were treated with different doses of inactive PARGi (PDD 00031705) for 72 hr. Cell viability was determined by the CellTiter-Glo assay. ( d ) Top: HeLa WT cells and PARG KO cells were treated with different doses of PARGi for 72 hr. Cell viability was determined by the CellTiter-Glo assay. Bottom: a schematic overview of 5 PARG isoforms based on Uniprot database annotation. The gRNAs targeting regions, the C-terminal catalytic domain, the catalytic inactive mutation, and the PCNA-interaction motif were indicated. The gRNAs used in HEK293A and HeLa cells were indicated. gRNA#1 used in HEK293A cells targets full-length PARG (i.e. isoform 1) and also isoforms 2 and 3, which are considered catalytically active isoforms according to the Uniport databse. gRNA#2 used in HeLa cells targets all five isoforms, but isoforms 4 and 5 are considered catalytically inactive according to the Uniport database. ( e ) HEK293A WT, PARG KO, and PARG KO cells reconstituted with WT PARG or catalytic inactivation PARG cells were treated with 0.01% MMS for 30 min. Blotting with anti-PARG and anti-pADPr antibodies was conducted to confirm the expression of PARG and PARG activity. Figure 1—figure supplement 1—source data 1. Original file for the western blot analysis in . Figure 1—figure supplement 1—source data 2. PDF containing and original scans of the relevant western blot analysis (anti-actin, anti-pADPr and anti-PARG) with highlighted bands and sample labels.

Article Snippet: Antibodies against PARP1 (9532 S), PARG (66564 S, which mainly recognizes full-length isoform 1, but also weakly recognizes isoforms 2 and 3 (data not shown)), and XRCC1 (2735 S) were purchased from Cell Signaling Technology.

Techniques: Glo Assay, Mutagenesis, Expressing, Activity Assay, Western Blot

Journal: eLife

Article Title: DePARylation is critical for S phase progression and cell survival

doi: 10.7554/eLife.89303

Figure Lengend Snippet: ( 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 and . Figure 1—source data 3. PDF containing , and original scans of the relevant western blot analysis (anti-actin and anti-PARG) with highlighted bands and sample labels.

Article Snippet: Antibodies against PARP1 (9532 S), PARG (66564 S, which mainly recognizes full-length isoform 1, but also weakly recognizes isoforms 2 and 3 (data not shown)), and XRCC1 (2735 S) were purchased from Cell Signaling Technology.

Techniques: CRISPR, Glo Assay, Mutagenesis, Western Blot

Journal: eLife

Article Title: DePARylation is critical for S phase progression and cell survival

doi: 10.7554/eLife.89303

Figure Lengend Snippet: ( 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 total cell lysates were immunoblotted with the indicated antibodies. ( b ) The total cell lysates in were blotted with anti-pADPr antibody. ( c ) Results of clonogenic assays conducted using HEK293A WT, PARG KO, PARP1/2 DKO, and PARG/PARP1/2 TKO cells treated with PARGi (1 µM) or PARPi (2 µM) or the combination of PARGi and PARPi for 7 days. ( d ) Relative NAD +level in HEK293A WT and PARG KO cells with the indicated treatment for different times. PARGi, 10 µM; FK866, 10 nM. ( e ) Results of clonogenic assays conducted using HEK293A WT and PARG KO cells with the indicated treatment for 48 hr. ( f ) Results of clonogenic assays were conducted using HeLa WT and PARG KO cells treated with PARGi (1 µM) or the combination of PARGi and NAM (100 µM) or NMN (1 mM) for 7 days. Figure 2—figure supplement 1—source data 1. Original file for the western blot in . Figure 2—figure supplement 1—source data 2. PDF containing and original scans of the relevant western blot analysis with highlighted bands and sample labels. Figure 2—figure supplement 1—source data 3. Original file for the colony formation assay in . Figure 2—figure supplement 1—source data 4. PDF containing and original scans of the relevant colony formation assay with highlighted bands and sample labels.

Article Snippet: Antibodies against PARP1 (9532 S), PARG (66564 S, which mainly recognizes full-length isoform 1, but also weakly recognizes isoforms 2 and 3 (data not shown)), and XRCC1 (2735 S) were purchased from Cell Signaling Technology.

Techniques: Western Blot, Colony Assay

Journal: eLife

Article Title: DePARylation is critical for S phase progression and cell survival

doi: 10.7554/eLife.89303

Figure Lengend Snippet: ( 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 and . Figure 2—source data 2. PDF containing and and original scans of the relevant western blot analysis and colony formation assay with highlighted bands and sample labels.

Article Snippet: Antibodies against PARP1 (9532 S), PARG (66564 S, which mainly recognizes full-length isoform 1, but also weakly recognizes isoforms 2 and 3 (data not shown)), and XRCC1 (2735 S) were purchased from Cell Signaling Technology.

Techniques: Staining, Glo Assay, Western Blot, Colony Assay

Journal: eLife

Article Title: DePARylation is critical for S phase progression and cell survival

doi: 10.7554/eLife.89303

Figure Lengend Snippet: ( 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 ) HEK293A WT and HEK293A PARG KO cells were mock-treated or pre-treated with 2 µM emetine for 90 min and then treated with PARGi for an additional 4 hr. Cells were fixed and stained with anti-pADPr antibody and PI. ( c ) HEK293A PARG KO cells were synchronized with double thymidine block (DTB). Cells remained with DTB or were released from DTB, treated with 10 µM PARGi for 4 hr, and then fixed and stained with anti-pADPr antibody and PI. ( d ) Representative images and results (left) of clonogenic assays conducted using control cells and DTB synchronized or released HEK293A PARG KO cells treated with the indicated doses of PARGi for 7 days, and quantification of crystal violet staining assay (right). ( e ) Results of clonogenic assays were conducted in PARG KO cells with indicated treatment for 7 days (PARGi, 1 µM; adarotene, 200 nM; CD437, 800 nM). Figure 3—source data 1. Original file for the colony formation assay in . Figure 3—source data 2. PDF containing and original scans of the relevant colony formation assay with highlighted bands and sample labels.

Article Snippet: Antibodies against PARP1 (9532 S), PARG (66564 S, which mainly recognizes full-length isoform 1, but also weakly recognizes isoforms 2 and 3 (data not shown)), and XRCC1 (2735 S) were purchased from Cell Signaling Technology.

Techniques: Staining, Blocking Assay, Control, Colony Assay

Journal: eLife

Article Title: DePARylation is critical for S phase progression and cell survival

doi: 10.7554/eLife.89303

Figure Lengend Snippet: ( 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 antibody (bottom panel). ( b ) Flow cytometry analysis of pADPr signaling in PARG KO cells which were pre-treated with DMSO, adarotene (200 nM) or CD437 (800 nM) for 16 hr, then 10 µM PARGi for another 4 hr.

Article Snippet: Antibodies against PARP1 (9532 S), PARG (66564 S, which mainly recognizes full-length isoform 1, but also weakly recognizes isoforms 2 and 3 (data not shown)), and XRCC1 (2735 S) were purchased from Cell Signaling Technology.

Techniques: Staining, Flow Cytometry

Journal: eLife

Article Title: DePARylation is critical for S phase progression and cell survival

doi: 10.7554/eLife.89303

Figure Lengend Snippet: ( 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 trapping-deficient mutant (del.p119K120S), were treated with PARGi (10 µM) for 4 hr. The total cell lysates and PARylated proteins enriched by Af1521 beads were immunoblotted with the indicated antibodies. ( c ) Alkaline comet assay results of HEK293A WT and PARG KO with PARGi (10 µM) treatment for different times. The comet-tail moments from 100 cells in each condition were measured and are shown in the box plot. The center line indicates the median, the box bounds indicate the first and third quartiles, and the whiskers indicate the maximum and minimum. ( d ) Flow cytometry analysis of pADPr signaling in HeLa WT and PARG KO cells treated with PARGi (10 µM) for different times. ( e ) PARG and PARP1 expression levels in HEK293A and HeLa cells. ( f ) HeLa WT and PARG KO cells were treated with PARGi (10 µM) for different times or for an additional 30 min with 0.01% MMS. The total cell lysates were immunoblotted with the indicated antibodies. Figure 4—figure supplement 1—source data 1. Original file for the western blot analysis in . Figure 4—figure supplement 1—source data 2. PDF containing and original scans of the relevant western blot analysis with highlighted bands and sample labels. Figure 4—figure supplement 1—source data 3. Original images for alkaline comet assay in .

Article Snippet: Antibodies against PARP1 (9532 S), PARG (66564 S, which mainly recognizes full-length isoform 1, but also weakly recognizes isoforms 2 and 3 (data not shown)), and XRCC1 (2735 S) were purchased from Cell Signaling Technology.

Techniques: Western Blot, Knock-Out, Mutagenesis, Alkaline Single Cell Gel Electrophoresis, Flow Cytometry, Expressing

Journal: eLife

Article Title: DePARylation is critical for S phase progression and cell survival

doi: 10.7554/eLife.89303

Figure Lengend Snippet: ( 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—source data 2. PDF containing and original scans of the relevant western blot analysis with highlighted bands and sample labels.

Article Snippet: Antibodies against PARP1 (9532 S), PARG (66564 S, which mainly recognizes full-length isoform 1, but also weakly recognizes isoforms 2 and 3 (data not shown)), and XRCC1 (2735 S) were purchased from Cell Signaling Technology.

Techniques: Western Blot, Glo Assay, Staining

Journal: eLife

Article Title: DePARylation is critical for S phase progression and cell survival

doi: 10.7554/eLife.89303

Figure Lengend Snippet: ( 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 antibody and sorted with flow cytometry. Cells with strong signals (top 25%, TOP) and weak signals (bottom 25%, BOT) were selected. The sgRNAs from these cells were then sequenced and analyzed. For cell viability screening, cells were treated with or without PARGi for 21 days before collection. ( b ) Scatter plot of DrugZ scores of PARG KO cells treated with or without PARGi treatment. The genes in the same pathway were marked with specific colors. A positive score indicates an enhanced pADPr signal, while a minus score indicates a decreased pADPr signal. ( c and d ) Ranking of PARGi co-essential genes on the basis of a DrugZ analysis of the results of CRISPR/Cas9 screens performed with Toronto Knock Out Library (version 3) in HEK293A PARG KO cells and HEK239A cells. ( e ) Analysis of biological processes of PARGi co-essential genes identified in HEK293A PARG KO cells and HEK239A cells. ( f ) HEK293A WT cells, PARG KO, POLB KO, LIG1 KO, XRCC1 KO, and LIG3 knockdown cells were treated with different doses of PARGi for 72 hr. Cell viability was determined by the CellTiter-Glo assay. ( g ) The cell Viability of HEK293A WT and PARG cells under POLB knockdown to PARGi. Cells were treated with different doses of PARGi for 72 hr. Figure 5—source data 1. NormZ score of FACS-based TKOv3 library screen conducted with PARG KO cells with or without PARGi. 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.

Article Snippet: Antibodies against PARP1 (9532 S), PARG (66564 S, which mainly recognizes full-length isoform 1, but also weakly recognizes isoforms 2 and 3 (data not shown)), and XRCC1 (2735 S) were purchased from Cell Signaling Technology.

Techniques: CRISPR, Selection, Staining, Flow Cytometry, Knock-Out, Knockdown, Glo Assay

Journal: eLife

Article Title: DePARylation is critical for S phase progression and cell survival

doi: 10.7554/eLife.89303

Figure Lengend Snippet: ( 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 increased sensitivity to PARGi in PARG KO cells. ARH3 or HPF knockout was confirmed by western blotting and DNA sequencing. Cell viability was measured with the CellTiter-Glo assay after 3 days. ( d ) XRCC1, LIG1, and POLB knockout in HEK293A was confirmed by western blotting and DNA sequencing. LIG3 knockdown was validated by western blotting, in which XRCC1 KO cells were included as controls. ( e ) Flow cytometry analysis of pADPr signaling in cells treated with PARGi (10 µM) for 4 hr. PARG KO, LIG1 KO, and LIG3 KD cells treated with PARGi showed S-phase-specific pAPDr signaling, while XRCC1 KO and POLB KO cells showed pAPDr signaling throughout the cell cycle. ( f ) POLB was knocked down by shRNA in both HEK293A WT and PARG KO cells. Figure 5—figure supplement 1—source data 1. NormZ score of FACS-based TKOv3 library screen conducted with HEK293A cells treated with PARGi. Figure 5—figure supplement 1—source data 2. Original file for the western blot analysis in . Figure 5—figure supplement 1—source data 3. PDF containing and original scans of the relevant western blot analysis with highlighted bands and sample labels.

Article Snippet: Antibodies against PARP1 (9532 S), PARG (66564 S, which mainly recognizes full-length isoform 1, but also weakly recognizes isoforms 2 and 3 (data not shown)), and XRCC1 (2735 S) were purchased from Cell Signaling Technology.

Techniques: Knock-Out, Western Blot, DNA Sequencing, Glo Assay, Knockdown, Flow Cytometry, shRNA

Journal: eLife

Article Title: DePARylation is critical for S phase progression and cell survival

doi: 10.7554/eLife.89303

Figure Lengend Snippet: ( 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 ) HEK293A cKO cells were validated by DNA sequencing. gDNA sequences are indicated at the top. The WT reference sequence was provided. ( c ) The dePARylation activity was assessed in whole cell lysates (WCLs) prepared from control wild-type (WT), PARG KO, and PARG cKO HEK293A cells. The dePARylation activity was measured with remnant pADPr recognized by anti-pADPr antibody, and normalized with the Control and the Blank samples as indicated in Materials and methods. n=4 independent measurements. ( d ) The PARG dePARylation activity in WCL prepared from control WT and PARG KO HEK293A was inhibited in a dose-dependent manner in the presence of PARGi. WCL prepared from PARG cKO cells was included as a control. n=3 independent measurements. ( e ) The PARG dePARylation activity shown in ( d ) is presented together with PARGi concentrations. Figure 7—figure supplement 2—source data 1. Original file for the western blot in . Figure 7—figure supplement 2—source data 2. PDF containing and original scans of the relevant western blot analysis with highlighted bands and sample labels.

Article Snippet: Antibodies against PARP1 (9532 S), PARG (66564 S, which mainly recognizes full-length isoform 1, but also weakly recognizes isoforms 2 and 3 (data not shown)), and XRCC1 (2735 S) were purchased from Cell Signaling Technology.

Techniques: Western Blot, DNA Sequencing, Sequencing, Activity Assay, Control

Journal: eLife

Article Title: DePARylation is critical for S phase progression and cell survival

doi: 10.7554/eLife.89303

Figure Lengend Snippet: ( 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—source data 2. PDF containing and original scans of the relevant western blot analysis and colony formation assay with highlighted bands and sample labels.

Article Snippet: Antibodies against PARP1 (9532 S), PARG (66564 S, which mainly recognizes full-length isoform 1, but also weakly recognizes isoforms 2 and 3 (data not shown)), and XRCC1 (2735 S) were purchased from Cell Signaling Technology.

Techniques: Derivative Assay, Knock-Out, Western Blot, Cell Culture, Clonogenic Assay, Clonogenic Cell Survival Assay, Mutagenesis, Blocking Assay, Staining, Colony Assay

Journal: Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology

Article Title: The cardioprotective effect of brief acidic reperfusion after ischemia in perfused rat hearts is not mimicked by inhibition of the Na(+)/H(+) exchanger NHE1.

doi: 10.1159/000331709

Figure Lengend Snippet: Fig. 5. Simulated ischemia and reperfusion induces pH- and NHE1-independent activation of Akt and JNK1/2 but not of p38 MAPK in HL- 1 cardiomyocytes. Cells were exposed to 5 h simulated ischemia followed by 15 min of acidic (pHo 6.0) or pH- neutral (pHo 7.4) reperfusion before another 1 h 45 min of pH-neutral reperfusion. During reperfusion cells were treated with or without 5 μM EIPA. Cells were lysed and kinase activation assessed by Western blotting using phospho- and total antibodies against a: Akt, b: JNK1, c JNK2, and d: p38 MAPK. Top a-d: representative Western blots. Bottom a-d: summary data for 3-4 experiments per condition, showing phospho- relative to total kinase abundance, normalized to normoxic control (means + SEM). Student’s t-test was performed. *p < 0.05 and **p < 0.01, indicates a significant difference from that in parallel normoxic controls.

Article Snippet: Antibodies against caspase-3 (detecting full-length caspase-3 (35 kDa) and the 17/19 kDa fragment resulting from cleavage at Asp175), poly (ADP-ribose) polymerase isoform 1 (PARP1) (detecting full length PARP1 (116 kDa) as well as the 89 kDa cleaved fragment), total JNK1/2, JNK1/2 phosphorylated at Thr183 and Tyr185, p38 MAPK, p38 MAPK phosphorylated at Thr180 and Tyr182, total Akt and Akt phosphorylated at Ser473 were from Cell Signaling Inc. 2,3,5-triphenyltetrazolium chloride (TTC) was from Sigma.

Techniques: Activation Assay, Western Blot, Control