Journal: Scientific Reports

Article Title: Nucleolar-nucleoplasmic shuttling of TARG1 and its control by DNA damage-induced poly-ADP-ribosylation and by nucleolar transcription

doi: 10.1038/s41598-018-25137-w

Figure Lengend Snippet: Tandem affinity purification of TARG1-containing protein complexes suggests functions of TARG1 in RNA biology. ( a ) Left panel: HEK293 Flp-In™ T-REx™ cells expressing TAP-TARG1 or the TAP-tag alone after doxycycline induction were lysed in the presence or absence of olaparib to inhibit ARTD1/2 activation during cell lysis. TAP-tag containing complexes were purified in two sequential affinity purification steps. Co-purified proteins were analyzed by LC-MS/MS and relative enrichment of TAP-TARG1 over TAP-tag control was determined by label-free quantitation (LFQ). Upper right panel: Domain structure of TAP-TARG1 and TAP constructs. Lower right panel: Fractions of the input lysate, the IgG and the CaM pulldown were analyzed for TARG1 by Western blot using mAb 3A5 (for characterization of TARG1 antibodies see Supplementary Fig. ). ( b ) Venn diagrams depicting the overlap between proteins associated with TARG1 in proteomics screens conducted in the absence (three biological replicates, p ≤ 0.01) or presence (two biological replicates, p ≤ 0.01) of olaparib in the lysis buffer with a fold enrichment of ≥2 (upper diagram) or ≥10 (lower diagram) over TAP control. ( c ) Fold enrichment of all proteins identified as TARG1 interacting proteins (p ≤ 0.01, ≥2-fold enrichment over TAP control) both in the absence and presence of olaparib. Only a fraction of grouped bars is specified with the corresponding protein names on the x-axis for clarity reasons. For details on the identified proteins see Supplementary Table , Supplementary Datasets and . ( d ) HEK293 cells were transfected with plasmids encoding for FLAG-TARG1 wildtype (WT) or ADPr binding-deficient FLAG-TARG1-G123E (GE). Presence of SSRP1, XRCC1 and CHD1L in FLAG immunoprecipitates (IP) and whole cell lysates (WCL) was analyzed by Western blotting. ( e ) HEK293 cells transiently expressing FLAG-TARG1 were lysed in TAP lysis buffer ±10 µM olaparib. Presence of ARTD1, XRCC1, CHD1L, KU80 and TARG1 after IP of FLAG-TARG1 was analyzed by Western blotting. ( f ) HEK293T cells transiently expressing FLAG-TARG1 were lysed in the presence of 10 µM olaparib. Presence of ARTD1, NCL, RPL7A and USP10 after IP against FLAG-TARG1 was analyzed by Western blotting. CHD1L was detected to control for inhibition of lysis-induced PARylation. ( d–f ) Full-length blots are presented in Supplementary Fig. .

Article Snippet: For antibody supershift experiments, 500 ng of an anti-TARG1 antibody (polyclonal, Eurogentec) were added prior to addition of the labeled RNA.

Techniques: Affinity Purification, Expressing, Activation Assay, Lysis, Purification, Liquid Chromatography with Mass Spectroscopy, Quantitation Assay, Construct, Western Blot, Transfection, Binding Assay, Inhibition

Journal: Scientific Reports

Article Title: Nucleolar-nucleoplasmic shuttling of TARG1 and its control by DNA damage-induced poly-ADP-ribosylation and by nucleolar transcription

doi: 10.1038/s41598-018-25137-w

Figure Lengend Snippet: OARD1 knock-out does not affect cell proliferation of HeLa cells. ( a ) TARG1 expression was analyzed in whole cell lysates of HeLa cells (WT) and the HeLa OARD1 −/− clones 3 and 12 by Western blotting using the TARG1-specific monoclonal antibody 3A5. α-Tubulin was detected as loading control. ( b ) Cell proliferation of HeLa cells and HeLa OARD1 −/− clones 3 and 12 was measured by counting living cells over 7 days. ( c ) HeLa OARD1 −/− (cl. 3) cells were stably transduced using a lentiviral vector allowing for doxycycline-inducible re-expression of HA-TARG1 or HA-TARG1-G43E. TARG1 protein expression was analyzed in HeLa OARD1 −/− HA-TARG1 wildtype or -G43E cells that were treated with 100 ng/ml doxycycline or were left untreated for 48 h by Western blotting. α-Tubulin levels were detected as a loading control. ( d ) Cell proliferation of HeLa OARD1 −/− HA-TARG1 or HA-TARG1-G43E cells, which were treated with 100 ng/ml doxycycline every 48–72 h or were left untreated (±Dox), was measured over 7 days by counting living cells. ( a and c ) Full-length blots are presented in Supplementary Fig. .

Article Snippet: For antibody supershift experiments, 500 ng of an anti-TARG1 antibody (polyclonal, Eurogentec) were added prior to addition of the labeled RNA.

Techniques: Knock-Out, Expressing, Clone Assay, Western Blot, Stable Transfection, Plasmid Preparation

Journal: Scientific Reports

Article Title: Nucleolar-nucleoplasmic shuttling of TARG1 and its control by DNA damage-induced poly-ADP-ribosylation and by nucleolar transcription

doi: 10.1038/s41598-018-25137-w

Figure Lengend Snippet: EGFP-TARG1 localizes to transcriptionally active nucleoli independent of ADP-ribosylation. ( a ) Live cell imaging of U2OS cells transiently expressing EGFP-TARG1 or EGFP together with mCherry-H2B. Intensity profiles for EGFP and mCherry fluorescence signals are displayed that were measured along the arrows depicted in the merge pictures. DIC: differential interference contrast. ( b ) Live cell imaging of U2OS cells transiently expressing EGFP-TARG1 wildtype (WT), G43E, I44E or G123E together with mCherry-H2B. For intensity profiles, see Supplementary Figures . ( c ) U2OS cells transiently expressing EGFP-TARG1 or EGFP-TARG1-G123E together with mCherry-H2B were treated with 10 ng/ml actinomycin D (ActD) or DMSO for 90 min. Subcellular localization of EGFP-TARG1 (wildtype or G123E) was analyzed by confocal microscopy in living cells.

Article Snippet: For antibody supershift experiments, 500 ng of an anti-TARG1 antibody (polyclonal, Eurogentec) were added prior to addition of the labeled RNA.

Techniques: Live Cell Imaging, Expressing, Fluorescence, Confocal Microscopy

Journal: Scientific Reports

Article Title: Nucleolar-nucleoplasmic shuttling of TARG1 and its control by DNA damage-induced poly-ADP-ribosylation and by nucleolar transcription

doi: 10.1038/s41598-018-25137-w

Figure Lengend Snippet: EGFP-TARG1 rapidly shuttles between the nucleoplasmic and the nucleolar compartment. ( a ) Fluorescence loss in photo-bleaching (FLIP) experiments in U2OS cells transiently expressing EGFP-TARG1, EGFP-TARG1-G123E or EGFP. mCherry-H2B was co-expressed to define the nucleoplasm (not shown). Representative images are shown. ( b–d ) EGFP fluorescence intensities of EGFP-TARG1 (upper panels), EGFP-TARG1-G123E (middle panels) and EGFP (lower panels) were measured in each ROI during photo-bleaching of nucleoplasmic EGFP ( b ), after total photo-bleaching of nucleoplasmic EGFP ( c ) or during photo-bleaching of cytoplasmic EGFP ( d ). Fluorescence intensities were normalized to the mean EGFP fluorescence intensity in the same ROI before bleaching and are expressed as mean ± SD of 7–12 cells as indicated.

Article Snippet: For antibody supershift experiments, 500 ng of an anti-TARG1 antibody (polyclonal, Eurogentec) were added prior to addition of the labeled RNA.

Techniques: Fluorescence, Expressing

Journal: Scientific Reports

Article Title: Nucleolar-nucleoplasmic shuttling of TARG1 and its control by DNA damage-induced poly-ADP-ribosylation and by nucleolar transcription

doi: 10.1038/s41598-018-25137-w

Figure Lengend Snippet: Nucleolar accumulation of TARG1 is lost upon H 2 O 2 -treatment. ( a ) U2OS cells transiently expressing EGFP-TARG1 or EGFP-TARG1-G123E together with mCherry-H2B were or were not (ctrl) treated with 1 mM H 2 O 2 . Subcellular localization of EGFP-TARG1 (wildtype/G123E) before (−) and during the first 20 min after treatment was analyzed in living cells by confocal microscopy. A representative experiment is depicted. ( b ) Quantifications of nucleolar EGFP intensities from cells imaged during the experiment depicted in panel A are given. Nucleolar EGFP-intensities were measured using ImageJ, normalized to nuclear EGFP-intensities at each time point and to the normalized nucleolar EGFP intensity 1 min after treatment (mean ± SD of at least 3 cells). ( c ) U2OS cells transiently expressing EGFP-TARG1 wildtype together with mCherry-H2B were treated with DMSO or 10 µM olaparib for 2 h before treatment with 1 mM H 2 O 2 . Subcellular localization of EGFP-TARG1 before (−) and during the first 20 min after treatment was analyzed in living cells by confocal microscopy. A representative experiment is depicted. ( d ) Quantifications of nucleolar EGFP intensities from cells imaged during the experiment depicted in panel c are given. Intensities were calculated as in panel b.

Article Snippet: For antibody supershift experiments, 500 ng of an anti-TARG1 antibody (polyclonal, Eurogentec) were added prior to addition of the labeled RNA.

Techniques: Expressing, Confocal Microscopy

Journal: Scientific Reports

Article Title: Nucleolar-nucleoplasmic shuttling of TARG1 and its control by DNA damage-induced poly-ADP-ribosylation and by nucleolar transcription

doi: 10.1038/s41598-018-25137-w

Figure Lengend Snippet: TARG1 is an RNA binding macrodomain. ( a ) TARG1 wildtype, -G43E, -I44E and -G123E were expressed as N-terminal His 6 -tag fusion proteins in bacteria and purified by immobilized metal affinity chromatography (IMAC). ALY, fused to an N-terminal glutathione S-transferase (GST)-tag, was expressed in bacteria and purified by glutathione affinity chromatography. Aliquots of the eluates, together with a bovine serum albumin (BSA) standard, were separated on an SDS-gel and stained with Coomassie blue. ( b ) Electrophoretic mobility shift assay (EMSA) using RNA Pentaprobe oligonucleotides . Purified 32 P-labeled Pentaprobe 9 ( 32 P-PP9) was incubated with the indicated amounts of purified GST, GST-ALY, His-TARG1 wildtype (WT), -G123E or -G43E (2.5–20 pmol corresponding to 83.3–666 nM, respectively). Free RNA and RNA-protein complexes were separated on native 7% poly-acrylamide gels. Mobility shifts were analyzed by auto-radiography. ( c ) EMSA performed as described in panel b with 32 P-PP9 and His-TARG1 (5 pmol, 0.16 µM), His-TARG1 in complex with a polyclonal antibody raised against TARG1 (Eurogentec) or with antibody alone. ( d ) EMSA of 32 P-PP7 that was incubated with constant amounts of His-TARG1 (5 pmol, 0.16 µM) and increasing amounts of cellular RNA (5–150 pmol, corresponding to 0.16–5 µM, calculated per nucleotide). ( e ) Thermal shift assay of 2 µM His-TARG1 WT or -G123E together with increasing amounts of cellular RNA (5–100 µM, calculated per nucleotide). Melting temperatures were determined according to and are presented as ΔT M to H 2 O control (ctrl; mean ± SD of 3 experiments). ( f ) Thermal shift assay of 2 µM His-TARG1 WT or -G123E in the presence of constant amounts of cellular RNA (100 µM, calculated per nucleotide) together with increasing amounts of ADPr. Melting temperatures are expressed as ΔT M to H 2 O control (ctrl; mean ± SD of 3 experiments). ( g ) EMSA of 32 P-PP7 that was incubated with constant amounts of His-TARG1 WT or -G123E (5 pmol, 0.16 µM) and increasing amounts of purified PAR (0.5–10 pmol, calculated per ADPr unit, corresponding to 0.016–0.33 µM) or ADPr (50–200 pmol, corresponding to 1.66–6.66 µM).

Article Snippet: For antibody supershift experiments, 500 ng of an anti-TARG1 antibody (polyclonal, Eurogentec) were added prior to addition of the labeled RNA.

Techniques: RNA Binding Assay, Purification, Affinity Chromatography, SDS-Gel, Staining, Electrophoretic Mobility Shift Assay, Labeling, Incubation, Thermal Shift Assay

Journal: Scientific Reports

Article Title: Nucleolar-nucleoplasmic shuttling of TARG1 and its control by DNA damage-induced poly-ADP-ribosylation and by nucleolar transcription

doi: 10.1038/s41598-018-25137-w

Figure Lengend Snippet: Model of a dual function of TARG1 in both RNA- and PAR-regulated cellular processes. Under steady-state conditions, TARG1 accumulates in nucleoli but constantly shuttles between nucleoli and the nucleoplasm (1). Accumulation of TARG1 in nucleoli is mediated by direct interaction with ribosomal RNA or proteins (RP) or other nucleolar proteins (NP; 2). PARylation modulates the localization and interactome of TARG1. TARG1 re-locates to the nucleoplasm upon DNA damage-induced ARTD1/2-dependent PARylation (3). In nucleoli, TARG1 may contribute to ribosome biogenesis, e.g., by counteracting PARylation of RPs or controlling nucleolar PAR scaffolds. DNA damage-induced PARylation might serve to sequester TARG1 to the nucleoplasm to regulate PAR turnover at DNA damage sites (4). Hypothetical interactions are marked by grey arrows.

Article Snippet: For antibody supershift experiments, 500 ng of an anti-TARG1 antibody (polyclonal, Eurogentec) were added prior to addition of the labeled RNA.

Techniques:

Journal: bioRxiv

Article Title: Targeted DNA ADP-ribosylation triggers templated repair in bacteria and base mutagenesis in eukaryotes

doi: 10.1101/2024.11.17.623984

Figure Lengend Snippet: a , Reversion of ADP-ribosylation of ssDNA in human cells by the TARG1 protein. b , Experimental setup for introducing edits in the EMX1 gene in HEK293T cells using an oligonucleotide repair template (RT). c, Extent of templated recombination (top), indel formation (middle) or base mutagenesis (bottom) using EMX 1 sgRNA1 in HEK293T cells with TARG1 intact (WT) or disrupted (Δ TARG1 ). Bars and error bars represent the mean and SEM of three independent transient transfections without selection or sorting. d, Frequency of base substitutions across the sgRNA target in the absence of the oligonucleotide RT. Results are shown with DNA nicking by Cas9 intact (top) or disabled (bottom). e, Extent of base mutagenesis of the ADP-ribosylated thymine across 17 target sites in five genes. For d and e, bars and error bars represent the mean and SEM of three independent transient transfections without selection or sorting. f, Extent of base mutagenesis based on the relative location of the ADP-ribosylated thymine. Cumulative thymine base editing across 21 sgRNA targets, within 37 5′-TYTN-3′ motifs at positions 3-14 (Position 1 being at the PAM-distal end). Solid black lines represent the median, gray lines represent the quartiles. Each dot represents the mean of three independent transient transfections without selection or sorting for a given sgRNA. g, Relationship between the outcome of base mutagenesis and the DarT2 recognition sequence. Distributions were calculated for base mutations occurring at 33 DarT2 recognition motifs across 21 sgRNAs. Bars and error bars represent the mean and SEM of fraction of total values.

Article Snippet: S15.) with anti- TARG1 antibody (Fisher Scientific, Cat.# 25249-1-AP) and anti-beta-actin antibody (Life technologies, Cat.# MA5-15739-HRP) as the housekeeping control.

Techniques: Mutagenesis, Transfection, Selection, Sequencing

Journal: Nucleic Acids Research

Article Title: TARG1 protects against toxic DNA ADP-ribosylation

doi: 10.1093/nar/gkab771

Figure Lengend Snippet: TARG1 removes thymidine-linked ADP-ribose from DNA and confers resistance to DarT toxicity in bacteria. ( A ) Structural comparison between TaqDarG-macrodomain (orange) bound to ADP-ribose (red) and TARG1 (light blue) with a covalent lysyl-ADP-ribose linkage (dark blue). To the right is a detailed view of the DarG catalytic lysine 80 (orange sticks) and TARG1 catalytic lysine 84 (blue sticks). ( B ) UV detection of DarT ADP-ribosylated DNA oligonucleotide de-ADP-ribosylation reactions with TaqDarG-macrodomain and TARG1. ( C ) Bacterial DarT toxicity rescue assay in BL21 DE3 using pBAD DarT and pET encoding DarG-macrodomain WT, DarG-macrodomain K80A, TARG1 WT or TARG1 K84A. pBAD expression is controlled with glucose or arabinose and pET expression is controlled with IPTG.

Article Snippet: Plasmids used here include: pBAD33-V5-DarT ( Thermus aquaticus ), pET28a-6xHis-TARG1-WT, pET28a-6xHis-TARG1-K84A, pET28a-6xHis-DarG-WT-1–155aa ( Thermus aquaticus ), pET28a-6xHis-DarG-K80A-1–155aa ( Thermus aquaticus ), pLIX_403-GFP-DarT-WT ( Thermus aquaticus ), pLIX_403-GFP-DarT-E160A ( Thermus aquaticus ), pLX304-TARG1-WT, pLX304-TARG1-K84A. pLIX_403 and pLX304 were gifts from David Root (pLIX_403 Addgene: #41395, pLX304 Addgene: #25890)

Techniques: Rescue Assay, Expressing

Journal: Nucleic Acids Research

Article Title: TARG1 protects against toxic DNA ADP-ribosylation

doi: 10.1093/nar/gkab771

Figure Lengend Snippet: DarT induces the DDR in TARG1-deficient cells and limits DNA replication ( A ) Representative images of clonogenic survival in U-2 OS WT or TARG1 KO cells expressing pLIX_403 GFP-DarT WT or GFP-DarT E160A. pLIX_403 expression was controlled by doxycycline. ( B ) U-2 OS WT or TARG1 KO cells treated with HU (2 mM, 24 h), CPT (1 μM, 1 h), MMS (2 mM, 1 h), DarT WT (24 h), DarT E160A (24 h). Levels of DDR proteins KAP1 pS824, RPA2 pS4/8, γH2AX were analysed in response to the aforementioned genotoxins. ( C ) QIBC analysis of EdU incorporation in U-2 OS WT and TARG1 KO cells in response to the genotoxins found in Figure 2B. Prior to fixation, cells were treated with EdU (10 μM, 30 min). Red bar represents the mean EdU intensity for each population and each point represents the mean EdU intensity for an individual nuclei. ( D ) U-2 OS WT or TARG1 KO cells expressing either DarT WT or TARG1 KO for 24 h were pulse labeled with CldU followed by IdU for 25 min each, as indicated by the labeling protocol, top. IdU track lengths were measured for 100 fibers per condition. Statistical tests were performed using ANOVA where ns is not significant and **** is P < 0.0001. In the box plots, the box extends from the 25th (lower edge) to 75th (higher edge) percentiles and the line within the box represents the median. Whiskers extend to the minimum and maximum values recorded. Representative fiber images can be found in . ( E ) U-2 OS TARG1 KO cells expressed GFP-DarT for 24 h and were treated with EdU (10 μM, 30 mins) prior to pre-extraction and fixation. Following a Click-iT reaction to visualise EdU, cells were then immunostained for GFP and chromatin-bound RPA2. Scale bar 20 μm. ( F ) QIBC analysis of chromatin-bound RPA2 of U-2 OS TARG1 KO cells in response to the genotoxins found in Figure 2B. Blue bar represents the mean RPA2 intensity for each population and each point represents the mean chromatin-bound RPA2 intensity for an individual nuclei.

Article Snippet: Plasmids used here include: pBAD33-V5-DarT ( Thermus aquaticus ), pET28a-6xHis-TARG1-WT, pET28a-6xHis-TARG1-K84A, pET28a-6xHis-DarG-WT-1–155aa ( Thermus aquaticus ), pET28a-6xHis-DarG-K80A-1–155aa ( Thermus aquaticus ), pLIX_403-GFP-DarT-WT ( Thermus aquaticus ), pLIX_403-GFP-DarT-E160A ( Thermus aquaticus ), pLX304-TARG1-WT, pLX304-TARG1-K84A. pLIX_403 and pLX304 were gifts from David Root (pLIX_403 Addgene: #41395, pLX304 Addgene: #25890)

Techniques: Expressing, Labeling

Journal: Nucleic Acids Research

Article Title: TARG1 protects against toxic DNA ADP-ribosylation

doi: 10.1093/nar/gkab771

Figure Lengend Snippet: DarT induces the DDR at sites of DNA replication ( A ) Representative images of U-2 OS TARG1 KO cells expressing either GFP-DarT WT or E160A for 24 h were pre-extracted and immunostained for GFP, γH2AX and PCNA. Nuclear DNA was counterstained with DAPI. Scale bar 20 μm. ( B ) Representative images of U-2 OS TARG1 KO cells expressing either GFP-DarT WT or E160A for 24 h were pre-extracted and immunostained for GFP, RPA2 pS4/8 and PCNA. Nuclear DNA was counterstained with DAPI. Scale bar 20 μm. ( C ) Workflow for QIBC analysis. ( D ) QIBC analysis of asynchronous U-2 OS TARG1 KO cells expressing GFP-DarT WT for 24 h. Cells were pre-extracted, fixed and immunostained as in A. QIBC was used to record total DAPI intensity, mean PCNA intensity and mean γH2AX intensity per nucleus for >1000 cells. DAPI and PCNA intensities for individual cells were used to generate the scatter plot and γH2AX intensity was used to colour points. ( E ) QIBC analysis of γH2AX in TARG1 KO cells in response to the genotoxin treatments found in Figure . Blue bar represents the mean γH2AX intensity for each population and each point represents the mean γH2AX intensity for an individual nuclei. ( F ) U-2 OS TARG1 KO cells were treated and analysed as in (D) and immunostained as in (B). ( G ) QIBC analysis as in E and immunostained as in (B).

Article Snippet: Plasmids used here include: pBAD33-V5-DarT ( Thermus aquaticus ), pET28a-6xHis-TARG1-WT, pET28a-6xHis-TARG1-K84A, pET28a-6xHis-DarG-WT-1–155aa ( Thermus aquaticus ), pET28a-6xHis-DarG-K80A-1–155aa ( Thermus aquaticus ), pLIX_403-GFP-DarT-WT ( Thermus aquaticus ), pLIX_403-GFP-DarT-E160A ( Thermus aquaticus ), pLX304-TARG1-WT, pLX304-TARG1-K84A. pLIX_403 and pLX304 were gifts from David Root (pLIX_403 Addgene: #41395, pLX304 Addgene: #25890)

Techniques: Expressing

Journal: Nucleic Acids Research

Article Title: TARG1 protects against toxic DNA ADP-ribosylation

doi: 10.1093/nar/gkab771

Figure Lengend Snippet: DarT induces ADP-ribose foci in S-phase cells with no ADP-ribosylation detectable by western blot. ( A ) U-2 OS TARG1 KO cells treated with HU (2 mM, 24 h), CPT (1 μM, 1 h), MMS (2 mM, 1 h), DarT (24 h), DarT E160A (24 h). Levels of ADP-ribosylation were assessed using the CST poly/mono ADP-ribose antibody in response to the aforementioned genotoxins. ( B ) QIBC analysis of ADP-ribosylation in TARG1 KO cells in response to the genotoxin treatments in A. Blue bar represents the mean ADP-ribosylation intensity for each population and each point represents the mean ADP-ribosylation intensity for an individual nuclei. ( C ) Representative images of U-2 OS TARG1 KO cells expressing either GFP-DarT WT or E160A for 24 h or cells treated with MMS (2 mM, 1 h) were pre-extracted and immunostained for GFP, ADP-ribosylation, PCNA and nuclear DNA was counterstained with DAPI. Scale bar 20 μm. ( D ) Magnified view of the region enclosed by the white square in (B) for DarT and MMS-treated cells. Scale bar 20 μM. ( E ) QIBC analysis of asynchronous U-2 OS TARG1 KO cells expressing GFP-DarT WT for 24 h or treated with MMS (2 mM, 1 h). Cells were pre-extracted, fixed and immunostained as in (C). QIBC was used to record total DAPI intensity, mean PCNA intensity and mean ADP-ribosylation intensity per nucleus for >1000 cells. DAPI and PCNA intensities for individual cells were used to generate the scatter plot and ADP-ribosylation intensity was used to color points.

Article Snippet: Plasmids used here include: pBAD33-V5-DarT ( Thermus aquaticus ), pET28a-6xHis-TARG1-WT, pET28a-6xHis-TARG1-K84A, pET28a-6xHis-DarG-WT-1–155aa ( Thermus aquaticus ), pET28a-6xHis-DarG-K80A-1–155aa ( Thermus aquaticus ), pLIX_403-GFP-DarT-WT ( Thermus aquaticus ), pLIX_403-GFP-DarT-E160A ( Thermus aquaticus ), pLX304-TARG1-WT, pLX304-TARG1-K84A. pLIX_403 and pLX304 were gifts from David Root (pLIX_403 Addgene: #41395, pLX304 Addgene: #25890)

Techniques: Western Blot, Expressing

Journal: Nucleic Acids Research

Article Title: TARG1 protects against toxic DNA ADP-ribosylation

doi: 10.1093/nar/gkab771

Figure Lengend Snippet: Detection of thymidine-linked ADP-ribosylation in human genomic DNA. ( A ) U-2 OS TARG1 KO cells expressing GFP-DarT (24 h) or treated with MMS (2 mM, 1 h) were also treated with olaparib (10 μM, 24 h) or veliparib (10 μM 24 h). Levels of KAP1 pS824, RPA2 pS4/8, γH2AX and ADP-ribosylation were assessed. ( B ) Representative images of U-2 OS TARG1 KO cells treated as in (A). Cells were pre-extracted and immunostained for GFP, ADP-ribosylation and chromatin-bound RPA2. Scale bar 10 μm. ( C ) U-2 OS WT or TARG1 KO cells treated with HU (2 mM, 24 h), CPT (1 μM, 1 h), MMS (2 mM, 1 h), DarT WT (24 h), DarT E160A (24 h). Genomic DNA was extracted and dotted onto nitrocellulose membranes and immunoblotted for dsDNA or ADP-ribosylation using the CST poly/mono ADP-ribose antibody. ( D ) Genomic DNA from U-2 OS TARG1 KO cells expressing DarT was extracted as in (C). DNA was incubated with either 1 μM TaqDarG-macrodomain WT or TARG1 WT recombinant proteins in vitro. DNA was then immunoblotted as in (C).

Article Snippet: Plasmids used here include: pBAD33-V5-DarT ( Thermus aquaticus ), pET28a-6xHis-TARG1-WT, pET28a-6xHis-TARG1-K84A, pET28a-6xHis-DarG-WT-1–155aa ( Thermus aquaticus ), pET28a-6xHis-DarG-K80A-1–155aa ( Thermus aquaticus ), pLIX_403-GFP-DarT-WT ( Thermus aquaticus ), pLIX_403-GFP-DarT-E160A ( Thermus aquaticus ), pLX304-TARG1-WT, pLX304-TARG1-K84A. pLIX_403 and pLX304 were gifts from David Root (pLIX_403 Addgene: #41395, pLX304 Addgene: #25890)

Techniques: Expressing, Incubation, Recombinant, In Vitro

Journal: Scientific Reports

Article Title: TARG1 affects EGFR signaling through the regulation of RNA metabolism

doi: 10.1038/s41598-025-08010-5

Figure Lengend Snippet: TARG1 loss leads to impaired cell migration. ( a ) Representative images (left) of wound healing assays with WT and TARG1 KO cells immediately after gap generation (0 h) and 24 h after it (24 h) in the presence of 100 ng/ml h-EGF in serum free medium, ( b ) in 10% FBS containing medium, and ( c ) in serum free medium. Scale bar, 100 μm. Wound closure rate (right) was determined as the percentage of gap closure 24 h after would generation. Data are mean ± SEM of n ≥ 3 independent experiments. Asterisks indicate p-values obtained by multiple t-test Holm-Sidak method, with alpha = 0.05. (ns. Not significant; ** p < 0.01).

Article Snippet: After blocking at room temperature (RT), the membranes were incubated with the primary antibodies: anti-EGFR [EP38Y] antibody (ab52894 Abcam, Cambridge, UK, 1:000), anti-pEGFR [phospho Y1068] (ab32430, Abcam Cambridge, UK, 1:8000), anti-GAPDH antibody (PA1-16,777, Thermo Fisher Scientific Inc., 1:3000) and anti-TARG1 antibody (25249-1-AP, ChromoTek GmbH, Planegg-Martinsried, Germany, 1:2000) overnight at 4 °C.

Techniques: Migration

Journal: Scientific Reports

Article Title: TARG1 affects EGFR signaling through the regulation of RNA metabolism

doi: 10.1038/s41598-025-08010-5

Figure Lengend Snippet: EGFR protein level is reduced in the TARG1 mutants. ( a ) Representative Western blot of phosphorylated EGFR (pEGFR), total EGFR (EGFR) and GAPDH at the indicated time points following h-EGF (100 ng/ml) stimulation in WT and TARG1 KO cells. GAPDH served as loading control. ( b ) Quantification of the phosphorylated EGFR (pEGFR) levels at the indicated time points in WT and TARG1 KO cells; (asterisks represents the significant differences between the WT and TARG1 KO at each time point) and the total pEGFR was calculated by dividing the pEGFR signal with the GAPDH signal) ( c ) Quantification of the relative phospho-EGFR (we divided the pEGFR/EGFR signal and then this relative pEGFR signal we further divided by the GADPH signal) level in WT and TARG1 KO cells. ( d ) Representative Western blot of the total EGFR level in WT, TARG1 KO and TARG1 knock down (KD) cell lines from whole cell lysate. Quantification of EGFR levels ( e ) in WT and the TARG1 KO and ( f ) in WT and TARG1 KD. Data in ( b , c , e , f ) are mean ± SEM ( n ≥ 3). Asterisks indicate p -values obtained by multiple t-test Holm-Sidak method, with alpha = 0.05. (** p < 0.01; *** p < 0.001). Representative images of EGFR internalization dynamics by immunofluorescence experiments of EGFR in WT and TARG1 KO cells ( g ) 4 h after serum starvation and ( h ) after 30 min h-EGF (100 ng/ml) stimulation following the 4 h serum starvation. Scale bar, 20 μm.

Article Snippet: After blocking at room temperature (RT), the membranes were incubated with the primary antibodies: anti-EGFR [EP38Y] antibody (ab52894 Abcam, Cambridge, UK, 1:000), anti-pEGFR [phospho Y1068] (ab32430, Abcam Cambridge, UK, 1:8000), anti-GAPDH antibody (PA1-16,777, Thermo Fisher Scientific Inc., 1:3000) and anti-TARG1 antibody (25249-1-AP, ChromoTek GmbH, Planegg-Martinsried, Germany, 1:2000) overnight at 4 °C.

Techniques: Western Blot, Control, Knockdown, Immunofluorescence

Journal: Scientific Reports

Article Title: TARG1 affects EGFR signaling through the regulation of RNA metabolism

doi: 10.1038/s41598-025-08010-5

Figure Lengend Snippet: Changes in mRNA levels of EGFR and response genes (MYC, CCDN1) were revealed by qRT-PCR analysis. ( a ) EGFR, ( b ) MYC, ( c ) CCND1 mRNA level in WT and TARG1 KO cells after 24 h serum starvation (24 h starv.), and after 24 serum starvation followed by 5 h of 10% serum refeeding (5 h ref.). ( d ) EGFR, ( e ) MYC, ( f ) CCND1 mRNA levels in WT and TARG1 KO cells cultured in normal medium (black bars; C), following transcription block (medium grey bars; DRB for 12 h), following translation block (dark grey bars; CHX for 12 h), and following combined transcription and translation block (light grey bars; DRB + CHX for 12 h). Relative gene expression was calculated by subtracting the Ct value of the gene of interest from the Ct value of RPL27. Data are mean ± SEM (n ≥ 3). Asterisks indicate p -values obtained by two-sided two-sample unequal variance t-test. (ns. not significant; * p < 0.05, ** p < 0.01, **** p < 0.0001).

Article Snippet: After blocking at room temperature (RT), the membranes were incubated with the primary antibodies: anti-EGFR [EP38Y] antibody (ab52894 Abcam, Cambridge, UK, 1:000), anti-pEGFR [phospho Y1068] (ab32430, Abcam Cambridge, UK, 1:8000), anti-GAPDH antibody (PA1-16,777, Thermo Fisher Scientific Inc., 1:3000) and anti-TARG1 antibody (25249-1-AP, ChromoTek GmbH, Planegg-Martinsried, Germany, 1:2000) overnight at 4 °C.

Techniques: Quantitative RT-PCR, Cell Culture, Blocking Assay, Gene Expression

Journal: Scientific Reports

Article Title: TARG1 affects EGFR signaling through the regulation of RNA metabolism

doi: 10.1038/s41598-025-08010-5

Figure Lengend Snippet: The TARG1 loss altered nuclear-cytoplasmic RNA distribution and translation after serum stimulation. ( a ) Representative images of total RNA staining in WT, TARG1 KO and TARG1 KD cells after 24 h serum starvation (24 h starv.) and after 24 h serum starvation followed by 5 h serum refeeding (5 h ref.) Scale bar, 20 μm. ( b ) Nucleo-cytoplasmic RNA distributions in WT, TARG1 KO and TARG1 KD cell lines after 24 h serum starvation (24 h starv.) and after 24 h serum starvation followed by 5 h serum refeeding (5 h ref.). Nucleo-cytoplasmic RNA distribution was quantified as the ratio of cytoplasmic to nuclear RNA intensities in individual cells, with cell and nuclear outlines identified using CellProfiler. Data are mean ± SEM (n ≥ 200). Asterisks indicate p -values obtained by two-way ANOVA followed by Turkey’s multiple comparison (ns. Not significant; ** p < 0.01; **** p < 0.0001). ( c ) SUnSET assay showing puromycin incorporation level reflecting translation rate of WT, TARG1 KO and TARG1 KD cells. GAPDH was used as loading control.

Article Snippet: After blocking at room temperature (RT), the membranes were incubated with the primary antibodies: anti-EGFR [EP38Y] antibody (ab52894 Abcam, Cambridge, UK, 1:000), anti-pEGFR [phospho Y1068] (ab32430, Abcam Cambridge, UK, 1:8000), anti-GAPDH antibody (PA1-16,777, Thermo Fisher Scientific Inc., 1:3000) and anti-TARG1 antibody (25249-1-AP, ChromoTek GmbH, Planegg-Martinsried, Germany, 1:2000) overnight at 4 °C.

Techniques: Staining, Comparison, Control

Journal: Scientific Reports

Article Title: TARG1 affects EGFR signaling through the regulation of RNA metabolism

doi: 10.1038/s41598-025-08010-5

Figure Lengend Snippet: TARG1 KO cells have increased sensitivity to MEK inhibition. Cell viability assay of WT and TARG1 KO ( a , b ) or TARG1 KD ( c , d ) cells treated with 100 nM Rapamycin ( a , c ), and with the MEK1/2 inhibitor, 25 µM U0126 alone (U0126) or in combination with 100 nM Rapamycin (U0126 + Rapa) ( b , d ) for 6 days. The graphs show the relative viability normalized to the untreated samples of each genotype. Data are mean ± SEM of n ≥ 3 independent experiments. Asterisks indicate p -values obtained by multiple t-test Holm-Sidak method, with alpha = 0.05. (ns. Not significant; *** p < 0.001;**** p < 0.0001).

Article Snippet: After blocking at room temperature (RT), the membranes were incubated with the primary antibodies: anti-EGFR [EP38Y] antibody (ab52894 Abcam, Cambridge, UK, 1:000), anti-pEGFR [phospho Y1068] (ab32430, Abcam Cambridge, UK, 1:8000), anti-GAPDH antibody (PA1-16,777, Thermo Fisher Scientific Inc., 1:3000) and anti-TARG1 antibody (25249-1-AP, ChromoTek GmbH, Planegg-Martinsried, Germany, 1:2000) overnight at 4 °C.

Techniques: Inhibition, Viability Assay