Journal: European Journal of Nuclear Medicine and Molecular Imaging

Article Title: PET imaging of PARP expression using 68 Ga-labelled inhibitors

doi: 10.1007/s00259-023-06249-6

Figure Lengend Snippet: Design of 68 Ga-DOTA-Olaparib for PARP-1 binding. ( a ) Chemical structure of the PARP-1 inhibitor Olaparib. ( b ) Co-crystal structure of Olaparib bound to the PARP-1 catalytic domain (PDB ID: 5DS3). ( c ) Chemical structure of DOTA-Olaparib. ( d ) Molecular docking of DOTA-Olaparib to PARP-1. ( e ) H-bonding interactions between DOTA-Olaparib and PARP-1 residues

Article Snippet: The PARP-1 protein (human, 11,040-H08B, Recombinant (His Tag)) was from Sino Biological.

Techniques: Binding Assay

Journal: European Journal of Nuclear Medicine and Molecular Imaging

Article Title: PET imaging of PARP expression using 68 Ga-labelled inhibitors

doi: 10.1007/s00259-023-06249-6

Figure Lengend Snippet: Cell uptake of the three 68 Ga-labelled radiotracers. ( a ) Western blot analysis probing the expression of PARP-1 in A549 and SK-OV-3 cells. ( b ) Cell uptake of the 68 Ga-labelled radiotracers in SK-OV-3 and A549 cells after 2 h of incubation. ( c ) The SK-OV-3/A549 uptake ratios of the. 68 Ga-labelled radiotracers. ( d ) Confocal images of cells stained with FL-Olaparib (green, left) and DAPI (blue, middle) alone. Confocal image of cells stained with FL-Olaparib and DAPI (right). ( e ) Confocal images of cells stained with FL-Olaparib with a 100-fold excess of Olaparib (left) and DAPI (blue, middle) alone. Confocal image of cells stained with FL-Olaparib with DAPI and a 100-fold excess of Olaparib (right). ( f ) Confocal images of cells stained with FL-Olaparib with a 1000-fold excess of DOTA-Olaparib (left) and DAPI (blue, middle) alone. Confocal image of cells stained with FL-Olaparib with DAPI and a 1000-fold excess of DOTA-Olaparib (right)

Article Snippet: The PARP-1 protein (human, 11,040-H08B, Recombinant (His Tag)) was from Sino Biological.

Techniques: Western Blot, Expressing, Incubation, Staining

Journal: European Journal of Nuclear Medicine and Molecular Imaging

Article Title: PET imaging of PARP expression using 68 Ga-labelled inhibitors

doi: 10.1007/s00259-023-06249-6

Figure Lengend Snippet: Association of 68 Ga-DOTA-Olaparib signalling with PARP-1 expression at the tumor tissue level. ( a ) Autoradiography and ( b ) H&E staining of SK-OV-3 models injected with 68 Ga-DOTA-Olaparib. ( c ) Representative image showing PARP-1 immunohistochemical staining

Article Snippet: The PARP-1 protein (human, 11,040-H08B, Recombinant (His Tag)) was from Sino Biological.

Techniques: Expressing, Autoradiography, Staining, Injection, Immunohistochemical staining

Journal: Molecular medicine reports

Article Title: SRPK1‑siRNA suppresses K562 cell growth and induces apoptosis via the PARP‑caspase3 pathway.

doi: 10.3892/mmr.2017.8032

Figure Lengend Snippet: Figure 5. Western blot results showing the protein levels of caspase‑3, cleaved caspase‑3, PARP, cleaved PARP, p53 and Bcl‑2/Bax. β‑actin was used as an internal loading control. Data are representative of three separate experiments. *P<0.05, compared with control groups.

Article Snippet: The primary antibodies were partly from Cell Signaling Technology, Inc. (Beverly, MA, USA) including caspase‐3 (1:1,000), cleaved caspase‐3 (1:1,000), PARP (1:1,000), cleaved PARP (1:1,000), p53 (1:1,000), Bax (1:1,000), Bcl‐2 (1:1,000) while β-actin antibodies (1:500) were purchased from Wuhan Boster Biological Technology, Ltd. (Wuhan, China).

Techniques: Western Blot, Control

Journal: Molecular cell

Article Title: SPARCLE , a p53-induced lncRNA, controls apoptosis after genotoxic stress by promoting PARP-1 cleavage

doi: 10.1016/j.molcel.2022.01.001

Figure Lengend Snippet: (A) PARP-1 domains. FI-FIII are Zinc finger domains. Arrow shows the caspase-3/7 cleavage site.

Article Snippet: In vitro transcription 2 μg of linearized plasmid encoding full-length SPARCLE ( SPARCLE 3K ), the first 75 nt ( SPARCLE 75 ), 178 nt ( SPARCLE 178 ) or 275 nt ( SPARCLE 275 ) of SPARCLE sequence or the reverse sequence of SPARCLE 275 ( ELCRAPS ) were in vitro transcribed using the MEGAScript T7 transcription kit (AM1334, ThermoFisher Scientific) following the manufacturer’s directions, but with increased incubation time (6 hr). . PARP-1 in vitro cleavage 50 ng of human recombinant full-length PARP-1 protein (11040-H08B, Sino Biological) were incubated at 37°C for 10 min with 0.5 units of human recombinant cleaved Caspase 3 protein (ab52101, Abcam) and with the indicated amount of in vitro transcribed SPARCLE 75 , SPARCLE 178 , SPARCLE 275 or ELCRAPS .

Techniques:

Journal: Cell Communication and Signaling : CCS

Article Title: Poly(ADP-ribosyl)ation of acetyltransferase NAT10 by PARP1 is required for its nucleoplasmic translocation and function in response to DNA damage

doi: 10.1186/s12964-022-00932-1

Figure Lengend Snippet: PARP1 PARylates NAT10 at K1016, K1017, and K1020 both in vitro and in vivo. A Four GST-NAT10 deletion mutants (∆1–201, ∆202–488, ∆489–753, and ∆754–1025) and GST control were bacterially purified, and subjected to in vitro PARation assay in the presence of PARP1 and NAD + . The reaction samples were resolved by SDS-PAGE, and analyzed by immunoblotting analyses with anti-PAR and anti-GST antibodies. B In vitro PARylation assays were performed using purified GST-NAT10 deletion fragments in the presence or absence of recombinant PARP1 enzyme, NAD + , and Olaparib. PARylated NAT10 was detected with an anti-PAR antibody. C – E GST-NAT10 deletion fragments were subjected to in vitro PARation assay as described in A. F , G Purified GST-NAT10 754–1025 proteins (WT, K1016A, K1017A, D1018A, and K1020A, K3A) were subjected to in vitro PARation assays in the presence of PARP1 and NAD + . PARylation of NAT10 was detected by immunoblotting with an anti-PAR antibody. In G, K3A represents the combined mutation in all three residues (K1016, K1017, and K1020). H Alignment of the NAT10 protein sequence among different organisms. Asterisk (*) indicates the full conservation of the residues of NAT10 among different species. I , J MCF-7 cells were transfected with HA-NAT10 or HA-NAT10 K3A expression vector. After 48 h of transfection, cells were treated with or without 1 mM MMS for 2 h ( I ) or 6 Gy IR ( J ). Thereafter, IP and immunoblotting analyses were conducted with the indicated antibodies

Article Snippet: The purified GST-NAT10 fragment (1 μg) was incubated with 100 ng of recombinant full-length PARP1 (Origene, #TP710053) in a reaction buffer containing 100 mM Tris–HCl (pH 8.0), 10 mM MgCl 2 , 1 mM DTT, 4 ng/ml sonicated salmon sperm DNA (Invitrogen, #AM9680), and 300 μM nicotinamide adenine dinucleotide (NAD + ) at 37 °C for 30 min.

Techniques: In Vitro, In Vivo, Purification, SDS Page, Western Blot, Recombinant, Mutagenesis, Sequencing, Transfection, Expressing, Plasmid Preparation

Journal: Cell Communication and Signaling : CCS

Article Title: Poly(ADP-ribosyl)ation of acetyltransferase NAT10 by PARP1 is required for its nucleoplasmic translocation and function in response to DNA damage

doi: 10.1186/s12964-022-00932-1

Figure Lengend Snippet: PARylation of NAT10 by PARP1 regulates its nucleoplasmic translocation and co-localization with MORC2. A MCF-7 cells were transfected with plasmid DNAs encoding Flag-MORC2, HA-NAT10, or HA-NAT10 K3A. After 48 h of transfection, cells were treated with or without 1 mM MMS for 2 h or 6 Gy IR. IF staining was performed with an anti-Flag (green) or anti-HA (red) antibody. DNA was counterstained with DAPI (blue). Scale bar, 2.5 μm. The quantitative results of cells with NAT10 nucleoplasmic translocation are presented in the right panel. ** p < 0.01; NS , no significance. B , C MCF-7 cells were transfected with HA-NAT10 and Flag-MORC2. After 48 h of transfection, cells were pretreated with or without 10 μM Olaparib for 3 h, and then treated with or without 1 mM MMS for another 2 h ( B ) or 6 Gy IR ( C ). IF staining was performed with an anti-Flag (green) or anti-HA (red) antibody. DNA was counterstained with DAPI (blue). Scale bar, 2.5 μm. The quantitative results of cells with NAT10 nucleoplasmic translocation are displayed in the right panel. ** p < 0.01; *** p < 0.001. D , E PARP1-KO MCF-7 cells were transfected with HA-NAT10 and Flag-MORC2. After 48 h of transfection, cells were treated with or without 1 mM MMS for another 2 h ( D ) or 6 Gy IR ( E ). IF staining was performed with an anti-Flag (green) or anti-HA (red) antibody. DNA was counterstained with DAPI (blue). Scale bar, 2.5 μm. The quantitative results of cells with NAT10 nucleoplasmic translocation are displayed in the right panel. ** p < 0.01; *** p < 0.001. Arrows indicate the colocalization between MORC2 and NAT10

Article Snippet: The purified GST-NAT10 fragment (1 μg) was incubated with 100 ng of recombinant full-length PARP1 (Origene, #TP710053) in a reaction buffer containing 100 mM Tris–HCl (pH 8.0), 10 mM MgCl 2 , 1 mM DTT, 4 ng/ml sonicated salmon sperm DNA (Invitrogen, #AM9680), and 300 μM nicotinamide adenine dinucleotide (NAD + ) at 37 °C for 30 min.

Techniques: Translocation Assay, Transfection, Plasmid Preparation, Staining

Journal: Cell Communication and Signaling : CCS

Article Title: Poly(ADP-ribosyl)ation of acetyltransferase NAT10 by PARP1 is required for its nucleoplasmic translocation and function in response to DNA damage

doi: 10.1186/s12964-022-00932-1

Figure Lengend Snippet: PARylation of NAT10 by PARP1 regulates its interaction with MORC2. A HEK293T cells were transfected with the indicated expression vectors. After 48 h of transfection, cells were treated with or without 1 mM MMS for 2 h and subjected to IP and immunoblotting analyses with the indicated antibodies. B , C BT549 cells were transfected with plasmid DNAs encoding pCDH, HA-NAT10, or HA-NAT10 K3A. After 48 h of transfection, cells were treated with or without 1 mM MMS for 2 h ( B ) or 6 Gy IR ( C ). IP and immunoblotting analyses were performed with the indicated antibodies. D – F MCF-7 and BT549 cells were pretreated with or without 10 μM Olaparib for 3 h, and then treated with or without 1 mM MMS for another 2 h. The sequential IP and immunoblotting analyses were performed with the indicated antibodies. G – I MCF-7 and BT549 cells were pretreated with or without 10 μM Olaparib for 3 h, and then treated with or without 6 Gy IR. The sequential IP and immunoblotting analyses were performed with the indicated antibodies

Article Snippet: The purified GST-NAT10 fragment (1 μg) was incubated with 100 ng of recombinant full-length PARP1 (Origene, #TP710053) in a reaction buffer containing 100 mM Tris–HCl (pH 8.0), 10 mM MgCl 2 , 1 mM DTT, 4 ng/ml sonicated salmon sperm DNA (Invitrogen, #AM9680), and 300 μM nicotinamide adenine dinucleotide (NAD + ) at 37 °C for 30 min.

Techniques: Transfection, Expressing, Western Blot, Plasmid Preparation

Journal: Cell Communication and Signaling : CCS

Article Title: Poly(ADP-ribosyl)ation of acetyltransferase NAT10 by PARP1 is required for its nucleoplasmic translocation and function in response to DNA damage

doi: 10.1186/s12964-022-00932-1

Figure Lengend Snippet: PARylation of NAT10 by PARP1 regulates MORC2 acetylation in response to DNA damage. A , B NAT10-KO MCF-7 and BT549 cells were transfected with plasmid DNAs encoding pCDH, HA-NAT10, or HA-NAT10 K3A. After 48 h of transfection, cells were treated with or without 1 mM MMS for 2 h ( A ) or 6 Gy IR ( B ), and then subjected to IP and immunoblotting with the indicated antibodies

Article Snippet: The purified GST-NAT10 fragment (1 μg) was incubated with 100 ng of recombinant full-length PARP1 (Origene, #TP710053) in a reaction buffer containing 100 mM Tris–HCl (pH 8.0), 10 mM MgCl 2 , 1 mM DTT, 4 ng/ml sonicated salmon sperm DNA (Invitrogen, #AM9680), and 300 μM nicotinamide adenine dinucleotide (NAD + ) at 37 °C for 30 min.

Techniques: Transfection, Plasmid Preparation, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Poly(ADP-ribosyl)ation of acetyltransferase NAT10 by PARP1 is required for its nucleoplasmic translocation and function in response to DNA damage

doi: 10.1186/s12964-022-00932-1

Figure Lengend Snippet: DNA damage induces MORC2 K767Ac in a PARP1-dependent manner. A MCF-7 cells were pretreated with or without 10 μM ATM inhibitor (KU-55933), 10 μM ATR inhibitor (VE-821), 10 μM DNA-PKcs inhibitor (NU7441), and 10 μM PARP inhibitor (Olaparib) for 3 h, and then treated with 1 mM MMS for 1 h. IP and immunoblotting analyses were performed with the indicated antibodies. Positive controls for these inhibitors are shown in the input. B HEK293T cells stably expressing pCDH and Flag-MORC2 were pretreated with or without 10 μM Olaparib for 3 h, and then treated with 1 mM MMS for another 2 h or 6 Gy IR. IP and immunoblotting analyses were performed with the indicated antibodies. C MCF-7 and BT549 cells were pretreated with or without 10 μM Olaparib for 3 h, and then treated with 1 mM MMS for another 2 h or 6 Gy IR. IP and immunoblotting analyses were performed with the indicated antibodies. D WT and PARP1-KO MCF-7 cells were treated with or without 1 mM MMS for 2 h or 6 Gy IR. Thereafter, IP and immunoblotting analyses were carried out with the inidicated antibodies. E BT549 cells were transfected with siNC or two independent siRNA targeting PARP1 (siPARP1). After 48 h of transfection, cells were treated with or without 1 mM MMS for 2 h or 6 Gy IR. IP and immunoblotting analyses were subsequently performed with the indicated antibodies

Article Snippet: The purified GST-NAT10 fragment (1 μg) was incubated with 100 ng of recombinant full-length PARP1 (Origene, #TP710053) in a reaction buffer containing 100 mM Tris–HCl (pH 8.0), 10 mM MgCl 2 , 1 mM DTT, 4 ng/ml sonicated salmon sperm DNA (Invitrogen, #AM9680), and 300 μM nicotinamide adenine dinucleotide (NAD + ) at 37 °C for 30 min.

Techniques: Western Blot, Stable Transfection, Expressing, Transfection

Journal: Cell Communication and Signaling : CCS

Article Title: Poly(ADP-ribosyl)ation of acetyltransferase NAT10 by PARP1 is required for its nucleoplasmic translocation and function in response to DNA damage

doi: 10.1186/s12964-022-00932-1

Figure Lengend Snippet: PARylation of NAT10 by PARP1 is required for cell survival in response to DNA damage. A , B NAT10-KO MCF-7 and BT549 cells stably expressing pCDH, HA-NAT10 WT, or HA-NAT10 K3A were treated with increasing doses of MMS and then subjected to clonogenic survival assays. Representative images of survival colonies are displayed in A and the corresponding quantitative results are shown in B . C , D NAT10-KO MCF-7 and BT549 cells stably expressing pCDH, HA-NAT10 WT, or HA-NAT10 K3A were treated with or without 6 Gy IR, and then subjected to clonogenic survival assays. Representative images of survival colonies are displayed in C and the corresponding quantitative results are shown in D

Article Snippet: The purified GST-NAT10 fragment (1 μg) was incubated with 100 ng of recombinant full-length PARP1 (Origene, #TP710053) in a reaction buffer containing 100 mM Tris–HCl (pH 8.0), 10 mM MgCl 2 , 1 mM DTT, 4 ng/ml sonicated salmon sperm DNA (Invitrogen, #AM9680), and 300 μM nicotinamide adenine dinucleotide (NAD + ) at 37 °C for 30 min.

Techniques: Stable Transfection, Expressing

Journal: Cell Communication and Signaling : CCS

Article Title: Poly(ADP-ribosyl)ation of acetyltransferase NAT10 by PARP1 is required for its nucleoplasmic translocation and function in response to DNA damage

doi: 10.1186/s12964-022-00932-1

Figure Lengend Snippet: The proposed working model. Activated PARP1 after DNA damage catalyzes the PARylation of NAT10, which is required for the translocation of NAT10 from the nucleolus to the nucleoplasm. NAT10 relocalization increases its co-localization and interaction with its substrate, MORC2, thereby enhancing MORC2 K767Ac in response to DNA damage

Article Snippet: The purified GST-NAT10 fragment (1 μg) was incubated with 100 ng of recombinant full-length PARP1 (Origene, #TP710053) in a reaction buffer containing 100 mM Tris–HCl (pH 8.0), 10 mM MgCl 2 , 1 mM DTT, 4 ng/ml sonicated salmon sperm DNA (Invitrogen, #AM9680), and 300 μM nicotinamide adenine dinucleotide (NAD + ) at 37 °C for 30 min.

Techniques: Translocation Assay

Journal: Journal of Cell Science

Article Title: Local inhibition of rRNA transcription without nucleolar segregation after targeted ion irradiation of the nucleolus

doi: 10.1242/jcs.232181

Figure Lengend Snippet: Area of irradiated and unirradiated HeLa-Parp1 – CB-tagRFP cells. Cells on the left side of the dotted blue line were irradiated with 10 or 100 Cpp (orange and red target points, respectively) and allowed to recover for 1.5 h. For targeting, a single slice image of the Parp1 signal of the cells was taken (orange frame). Neighbouring cells on the right side of the blue dotted line were not irradiated and were used as controls. After 5EU incorporation and immunofluorescence staining, cells were imaged as z -stacks of 33 layers, with 300 nm distance in the single channels of Parp1, 5EU, γH2AX and phase contrast. Stacks were deconvolved for improved depth resolution. A single slice from the centre of the stack is shown. Arrows mark the target and hit areas verified by γH2AX. The best examples are the hit nucleoli in the three bottom cells (marked by asterisks). The outlines of these nucleoli in the phase contrast image still correlate with the outline before irradiation. The γH2AX foci are completely embedded in the nucleoli and at their sites; 5EU and Parp1 signals are less intense as in direct proximity. However, the irradiated nucleoli and the residual nucleoli of the corresponding nucleus do not show an overall decrease in 5EU signal. Fluctuations in brightness correlate with Parp1 fluctuations before irradiation and are also observable in control cells.

Article Snippet: A fluorescent, tagRFP-labelled chromobody (ChromoTek) marks the endogenous Parp1 protein in these cells.

Techniques: Irradiation, Immunofluorescence, Staining, Control

Journal: Journal of Cell Science

Article Title: Local inhibition of rRNA transcription without nucleolar segregation after targeted ion irradiation of the nucleolus

doi: 10.1242/jcs.232181

Figure Lengend Snippet: Certain nucleoli hits after irradiation with 1, 10 and 50 Cpp and their influence on rRNA transcription. Recovery time was between 30 min and 7 h. Target definition images before irradiation are shown on the left. γH2AX, 5EU, Parp1 and phase contrast images of single cells were taken in the focus depth of the γH2AX foci after IF staining. Additionally, intensity line plots through the γH2AX foci along the white dotted line of the merged γH2AX+5EU image are shown on the right. Contrast was chosen such that 0.1% of the pixels were in saturation. The intensity plots show how 5EU and Parp1 signals (red and blue, respectively) were locally reduced at the damage site marked by γH2AX (black). Two-dimensional location is marked by yellow arrows. In these images, this correlates with local reduction of Parp1 and at higher doses additionally with altered appearance of the nucleolus in the phase contrast image. For better cell reconstruction, we used tiled images that consisted of 2×2 fields of view from the camera before irradiation and 3×3 after IF staining; thus, some cell images show the edge of the tiles. Scale bars: 3 µm.

Article Snippet: A fluorescent, tagRFP-labelled chromobody (ChromoTek) marks the endogenous Parp1 protein in these cells.

Techniques: Irradiation, Staining

Journal: Journal of Cell Science

Article Title: Local inhibition of rRNA transcription without nucleolar segregation after targeted ion irradiation of the nucleolus

doi: 10.1242/jcs.232181

Figure Lengend Snippet: Cross-sections through z -stacks of certain nucleolus hit examples used for 5EU and Parp1 analysis. (A) 5EU and Parp1 signals were analysed in the central slice of the γH2AX foci (compare with Fig. 2 ). Nucleolar signal of 5EU is in green and γH2AX in red. The z -stacks shown consist of 13 slices with distance 300 nm, containing information of the single nucleus. (B) Analysis of single foci showing certain nucleolus hits on 5EU incorporation (left) and Parp1 distribution (right). Boxplots show median of the 5EU and Parp1 intensity ( I 5EU , I Parp1 ) ratios, in which the box represents the 2nd quartile, error bars show 1.5× the interquartile range and dots indicate outliers of the distribution. The signal intensity at the focus I …,focus minus the nucleus background signal I …,nucleus was calculated as a ratio of the signal intensity in the residual nucleolus I …,nucleolus minus the nucleus background signal I …,nucleus . 5EU and Parp1 signals were significantly reduced at the DNA damage site marked by γH2AX. A one-sample signed rank test (*) was used to test the hypothesis H 0 : median=1, which gave P <0.001. For the higher rates of 10 and 50 Cpp, 5EU incorporation and Parp1 intensity significantly decreased to about 0.2 and 0.5, respectively. The Mann–Whitney rank sum test was used to test the difference of the medians for 10 or 50 Cpp compared with 1 Cpp, which gave + P <0.001 for 5EU and ++ P <0.05 for Parp1. Scale bars: 3 µm (for all cross-sections).

Article Snippet: A fluorescent, tagRFP-labelled chromobody (ChromoTek) marks the endogenous Parp1 protein in these cells.

Techniques: MANN-WHITNEY

Journal: Journal of Cell Science

Article Title: Local inhibition of rRNA transcription without nucleolar segregation after targeted ion irradiation of the nucleolus

doi: 10.1242/jcs.232181

Figure Lengend Snippet: Analysis of single foci showing certain nucleolus hits on 5EU incorporation and Parp1 distribution. Frequency of single foci, showing certain nucleolus hits with dips in the 5EU and Parp1 signals. If the ratio was below 0.80, the area was defined as a dip. Error bars describe the 95% confidence Wilson score intervals of the binomial proportion.

Article Snippet: A fluorescent, tagRFP-labelled chromobody (ChromoTek) marks the endogenous Parp1 protein in these cells.

Techniques: