Journal: International Journal of Biological Sciences

Article Title: Poly (ADP-ribose) polymerase 1 (PARP1) inhibition promotes pulmonary metastasis of osteosarcoma by boosting ezrin phosphorylation

doi: 10.7150/ijbs.58784

Figure Lengend Snippet: The reduction of PARP1-mediated poly (ADP-ribose) enhanced the migratory and invasive properties of osteosarcoma cells. (A) Colony formation assay for detection of the proliferation of 143B and K7M2 cells treated with DMSO or olaparib for 48 h. (B) Migratory properties of 143B and K7M2 cells treated with DMSO or olaparib were tested using wound healing assays. Quantification of percentage of wound closure was determined by analysis using ImageJ software. Mean +/-S.D. of three independent experiments. Scale bars, 300 μm. (C) Invasive behaviors in collagen-type-IV gels of 143B and K7M2 cells with DMSO or olaparib were observed in chamber invasion assay. Scale bars, 100 μm. (D) The effect of PARP1 and PARP2 knockdown on the migration properties of 143B cells was tested via wound healing assay. Scale bars, 300 μm. (E) The effect of PARP1 and PARP2 knockdown on the invasive behavior of 143B cells was tested by chamber invasion assay. Scale bars, 100 μm. (F) Wound healing assay for detection of the migrative property for OS cells treated with DMSO, PARP inhibitors, respectively. (G) Analysis of transwell invasion assay for OS cells treated with DMSO, PARP inhibitors, respectively. (H) Represented images of the adhesion ability of 143B and K7M2 cells treated with DMSO or olaparib on fibronectin or collagen-type-I coated plates. Quantification of the adherent cells was determined by the OD value at 560 nm.

Article Snippet: His-tagged HSA protein and his-tagged PARP1 protein were purchased from Sino Biological Inc.

Techniques: Colony Assay, Software, Invasion Assay, Migration, Wound Healing Assay, Transwell Invasion Assay

Journal: International Journal of Biological Sciences

Article Title: Poly (ADP-ribose) polymerase 1 (PARP1) inhibition promotes pulmonary metastasis of osteosarcoma by boosting ezrin phosphorylation

doi: 10.7150/ijbs.58784

Figure Lengend Snippet: Olaparib induced OS migration and invasion were ezrin-dependent. (A) Human recombinant his-tagged PARP1 protein based pull-down assay was performed in OS cell lysates to identify PAPR1 interactive proteins. Human serum albumin (HSA) was used as control. The obtained proteins were separated in 10% SDS-PAGE gel flowed by silver staining. (B) co-IP assay for baiting PARP1 interactive proteins using anti-PARP1 antibody in OS cell lysates. IgG was used as negative control. The obtained proteins were separated in 10% SDS-PAGE gel flowed by silver staining. (C) 143B cell lysate was subjected for co-IP with anti-PARP1 or control IgG antibodies followed by western blotting with the indicated antibodies to verify the interaction between ezrin and PARP1 (n = 3). (D) Representative images from wound healing assay showing the effect of olaparib on cell migration of ezrin wildtype (ezrin WT ) and ezrin knock down (ezrin KD ) 143B cells. Wound spaces were analyzed using ImageJ. Scale bars, 300 μm. (E) Representative images from transwell assay showing the effect of olaparib on cell invasion of ezrin wildtype (ezrin WT ) and ezrin knock down (ezrin KD ) 143B cells. Invaded cells were counted in 15 random fields on the lower surface of the filters and expressed as ratio (fold) of invaded cells compared with the vehicle group. Scale bars, 300 μm. (F) Weight of NOD/SCID mice bearing orthotopic ezrin WT or ezrin KD 143B-mCherry tumors treated with olaparib or vehicle (mean ± s.e.m.; n = 8 mice per group). The statistical analyses were performed using one-way ANOVA with Tukey's multiple comparison tests. (G) Number of metastatic nodules per lung in NOD/SCID mice treated with olaparib or vehicle.

Article Snippet: His-tagged HSA protein and his-tagged PARP1 protein were purchased from Sino Biological Inc.

Techniques: Migration, Recombinant, Pull Down Assay, SDS Page, Silver Staining, Co-Immunoprecipitation Assay, Negative Control, Western Blot, Wound Healing Assay, Transwell Assay

Journal: International Journal of Biological Sciences

Article Title: Poly (ADP-ribose) polymerase 1 (PARP1) inhibition promotes pulmonary metastasis of osteosarcoma by boosting ezrin phosphorylation

doi: 10.7150/ijbs.58784

Figure Lengend Snippet: The PARylation of ezrin triggered by PARP1 hindered its phosphorylation. (A) siRNA induced PARP1 knock down and PARP enzymatic inhibition with olaparib both increased ezrin phosphorylation without affecting the total ezrin. The protein level of PAR, p-Ezrin, ezrin and PARP1 was determined using western blotting. β-actin was used as a loading control. (B) Olaparib promoted the phosphorylation of p-Ezrin independent on the kinases. (C) Binding domains of ezrin with PARP1 identified by co-IP using truncated versions of ezrin was verified by western blotting. (D) Molecular modeling of the interaction between ezrin and PARP1 interaction at full length. (E) Representative images from wound healing assay showing the effect of olaparib on cell migration of ezrin KD+WT and ezrin KD+Mut 143B cells. Wound spaces were analyzed using ImageJ. Scale bars, 300 μm. (F) Representative images from transwell assay showing the effect of olaparib on cell invasion of ezrin KD+WT and ezrin KD+Mut 143B cells. Invaded cells were counted in 15 random fields on the lower surface of the filters and expressed as ratio (fold) of invaded cells compared with the vehicle group.

Article Snippet: His-tagged HSA protein and his-tagged PARP1 protein were purchased from Sino Biological Inc.

Techniques: Inhibition, Western Blot, Binding Assay, Co-Immunoprecipitation Assay, Wound Healing Assay, Migration, Transwell Assay

Journal: Cell Reports Medicine

Article Title: FBXO3-mediated DUSP9 ubiquitination promotes leukemia stem cell maintenance and tyrosine kinase inhibitor resistance in chronic myeloid leukemia

doi: 10.1016/j.xcrm.2026.102686

Figure Lengend Snippet: FBXO3 deletion suppressed IM-sensitive/-resistant cells and primary CML CD34 + cells through PARP1-mediated endogenous apoptosis in vitro (A) CRISPR-Cas9-mediated FBXO3 knockout in K562/KBM5-T315I cells confirmed by western blot (left: representative blot; right: quantification, n = 3). Data are represented as mean ± SEM; t test; ∗∗∗∗ p < 0.0001. (B) Growth curves of control/sgFBXO3 cells over 7 days ( n = 3). Data are represented as mean ± SD; two-way ANOVA; ∗∗∗ p < 0.001. (C) Soft agar colony formation (≥50 cells/clone, scale bars: 100 μm). Data are represented as mean ± SEM; t test; ∗ p < 0.05, ∗∗ p < 0.01. (D and E) Enhanced apoptosis in sgFBXO3 cells: (D) annexin V/DAPI staining; (E) JC-10 mitochondrial depolarization. Data are represented as mean ± SEM; t test; ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; n = 3. (F) Western blot analysis of apoptosis-related proteins in control/sgFBXO3 cells. Data are represented as mean ± SEM; t test; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001; ns indicates no significance. (G) shRNA-mediated FBXO3 knockdown efficiency in CML/normal CD34 + cells. Data are represented as mean ± SEM; t test; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. (H) Colony formation assays in primary CML and normal CD34 + cell; scale bars, 100 μm, n = 3. Data are represented as mean ± SEM; t test; ∗∗ p < 0.01; ns indicates no significance. (I) Serial replating capacity of CD34 + cells (three generations). Data are represented as mean ± SEM; t test; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ns indicates no significance. (J and K) Apoptosis in FBXO3-knockdown CD34 + cells: (J) annexin V/DAPI ( n = 3); (K) annexin V/CFSE ( n = 3). Data are represented as mean ± SEM; t test; ∗ p < 0.05, ∗∗ p < 0.01; ns indicates no significance. (L and M) WB analysis of PARP1-mediated apoptotic proteins in CD34 + cells; n = 3. Data are represented as mean ± SEM; t test; ∗ p < 0.05, ∗∗ p < 0.01; ns indicates no significance.

Article Snippet: PARP1 , Proteintech , Cat#13371-1-AP; RRID: AB_2160459.

Techniques: In Vitro, CRISPR, Knock-Out, Western Blot, Control, Staining, shRNA, Knockdown

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: Fig. 2 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 MgCl2, 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, Control, 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: Fig. 3 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 MgCl2, 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: Fig. 4 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 MgCl2, 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: Fig. 6 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 MgCl2, 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: Fig. 5 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 MgCl2, 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: Fig. 7 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 MgCl2, 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: Fig. 8 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 MgCl2, 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: Scientific Reports

Article Title: A Novel Resveratrol Based Tubulin Inhibitor Induces Mitotic Arrest and Activates Apoptosis in Cancer Cells

doi: 10.1038/srep34653

Figure Lengend Snippet: A549 and CEM cells were treated with SS28 (5 μM) for 24, 48 and 72 h. Whole cell lysate was prepared and proteins were resolved on a SDS-PAGE and western blotting was performed using specific primary and secondary antibodies. Blots shown are representative blots of two independent experiments with identical results. Actin was used as the loading control. ( A ) For CEM cells p53, Ku70, Cyclin B1, Cdk6, PARP-1, Caspase 9 and Caspase 3 proteins were evaluated. ( B ) Quantification of the proteins shown in panel A is represented as bar diagram with error bars. ( C ) For A549 cells, p73, Ku80, Cdk6, Cyclin B1, Caspase 9 and Caspase 3 proteins were evaluated. ( D ) Quantification of the respective proteins is shown in bar diagram with error bars.

Article Snippet: Besides, we also observed activation of PARP1 upon treatment with SS28 in CEM cells.

Techniques: SDS Page, Western Blot, Control