Journal: Oncology Reports

Article Title: DEPP1: A prognostic biomarker linked to stroma-rich and immunosuppressive microenvironment, promoting oxaliplatin resistance in gastric cancer

doi: 10.3892/or.2025.8915

Figure Lengend Snippet: DEPP1 enhances oxaliplatin resistance in gastric cancer cells in vitro . Effects of oxaliplatin and fluorouracil supplementation on DEPP1 protein levels in (A) MKN45 and (B) HGC27 cells, respectively. Validation of DEPP1 overexpression in (C) MKN45 and (D) HGC27 cells. (E) Impact of DEPP1 overexpression on MKN45 cell proliferation, assessed using EdU flow cytometry. (F) Influence of DEPP1 overexpression on MKN45 proliferation following a 24-h treatment with 10 µM oxaliplatin. (G) Effects of ectopic DEPP1 expression on oxaliplatin-induced apoptosis (20 µM) in MKN45 cells, as measured by Annexin V/PI flow cytometry. (H) Analysis of full-length and cleaved PARP protein levels following forced DEPP1 expression in (H) MKN45 and (J) HGC27 cells. *P<0.05 and ***P<0.001 DEPP1, decidual protein induced by progesterone. ns, not significant.

Article Snippet: The membranes were then incubated with 5% skimmed milk at room temperature for 1 h. Following this, the membranes were probed with primary antibodies against DEPP1 (1:1,000; cat. no. 25833-1-AP; Proteintech Group, Inc.), PARP (1:1,000; cat. no. 9532; Cell Signaling Technology, Inc.), cleaved PARP (1:500; cat. no. HY- P80448 ; MedChemExpress), GAPDH (1:1,000; cat. no. 5174; Cell Signaling Technology, Inc.), or β-actin (1:20,000; cat. no. 66009-1-Ig; Proteintech Group, Inc.) at 4°C overnight, followed by incubation with peroxidase-conjugated goat anti-rabbit IgG (33101ES60, 1:5,000, Shanghai Yeasen Biotechnology Co., Ltd.) or peroxidase-conjugated goat anti-mouse IgG (1:5,000; cat. no. BL001A; Biosharp Life Sciences) at room temperature for 1 h. Finally, proteins were visualized using an Omni-ECLTMPico Light Chemiluminescence Kit (cat. no. SQ202L; Epizyme, Inc.).

Techniques: In Vitro, Biomarker Discovery, Over Expression, Flow Cytometry, Expressing

Journal: The Febs Journal

Article Title: ENPP 1 processes protein ADP ‐ribosylation in vitro

doi: 10.1111/febs.13811

Figure Lengend Snippet: Production of ENPP recombinant proteins. (A) Representative and schematic illustrations of recombinant mouse ENPP 2‐1‐Target (m ENPP 2‐1‐T) chimera purified as described in Kato et al . [42,43] and recombinant 8xHistidine tag version (m ENPP 2‐1‐8xHis). (B) Flow chart for the purification of m ENPP 2‐1‐T and m ENPP 2‐1‐8xHis. (C) Left panel, the purity of recombinant m ENPP 2‐1‐T and m ENPP 2‐1‐8xHis enzymes was analysed using separation of 20 μ m of protein on an SDS / PAGE gel followed by staining with Coomassie. Right panel, 20 μ m of m ENPP 2‐1‐T was probed by anti‐6xHis western blot. (D) 1 μg of m ENPP 2‐1‐T and m ENPP 2‐1‐8xHis enzymes were used as substrates for PNG ase F and Endo H deglycosylation enzymes. Samples were resolved on SDS / PAGE and stained by Coomassie. Black star indicates glycosylated ENPP 1, green star indicates deglycosylated ENPP 1, red star indicates deglycosylation resistant ENPP1.

Article Snippet: The 6xHis‐ hs PARP1 (wild‐type) was expressed and purified from E. coli , attached to MagneHis beads (Promega Corporation, Madison, WI, USA), and PARylated as described previously with the following changes: PARP1 (final concentration 1 μ m ) was autoPARylated in the presence of 1 m m β‐NAD + for 30 min at 37 °C.

Techniques: Recombinant, Purification, SDS Page, Staining, Western Blot

Journal: The Febs Journal

Article Title: ENPP 1 processes protein ADP ‐ribosylation in vitro

doi: 10.1111/febs.13811

Figure Lengend Snippet: ENPP 1 is able to hydrolyse protein poly( ADP ‐ribosyl)ation. About 70 n m of human recombinant PARP 1 was automodified to produce ~ 3 μ m PAR substrate (defined in monomeric ADP ‐ribose units) and incubated with buffer only (control) and decreasing concentrations of m ENPP 2‐1‐T (A), m ENPP 2‐1‐8xHis (B) and NUDT 16 (C). Samples were fractionated on SDS / PAGE and transferred on nitrocellulose membranes. Membranes were first stained with S‐Ponceau and then probed with anti‐ PAR antibody. (D) Time point hydrolysis of PAR ylated PARP 1 was performed at indicated concentrations and times with NUDT 16, m ENPP 2‐1‐T and SVP . Samples were resolved on SDS / PAGE and transferred on nitrocellulose membranes. Membranes were first stained with S‐Ponceau and then probed with anti‐ PAR antibody.

Article Snippet: The 6xHis‐ hs PARP1 (wild‐type) was expressed and purified from E. coli , attached to MagneHis beads (Promega Corporation, Madison, WI, USA), and PARylated as described previously with the following changes: PARP1 (final concentration 1 μ m ) was autoPARylated in the presence of 1 m m β‐NAD + for 30 min at 37 °C.

Techniques: Recombinant, Incubation, SDS Page, Staining

Journal: The Febs Journal

Article Title: ENPP 1 processes protein ADP ‐ribosylation in vitro

doi: 10.1111/febs.13811

Figure Lengend Snippet: ENPP 1 is able to hydrolyse protein poly‐ and mono‐( ADP ‐ribosyl)ation producing PRAMP and AMP . (A) Schematic illustration of protein ADP ‐ribosylation in the presence of NAD + labelled on the alpha phosphate group with 32 P. Enzymes used in experiments showed in panels B–D, and cleavable chemical bonds in radiolabelled MAR / PAR were indicated. Main reaction products of phosphodiesterases‐dependent hydrolysis of radiolabelled protein PAR ylation were represented. (B) Human recombinant PARP 1 was automodified in the presence of [ 32 P]‐ NAD + and then incubated with buffer (Control), 18 μ m of recombinant NUDT 16, 4 μ m of recombinant ENPP 2‐1‐T, 1 μ m of PARG and 0.45 μ m of purified SVP for 3 h at 30 °C. In top panel, samples were resolved on SDS / PAGE and [ 32 P]‐ NAD + incorporation was detected by autoradiography. In bottom panel, reactions described in top panel were loaded on TLC plate. (C) Top panel, 1 μ m of recombinant PARP 1‐E988Q mutant was automodified using 32 P‐labelled NAD + and then incubated with buffer only (control), 5 μ m of recombinant NUDT 16, 5 μ m of recombinant m ENPP 2‐1‐T or 2 μ m of purified SVP . Samples were resolved on SDS / PAGE and [ 32 P]‐ NAD + incorporation was detected by autoradiography. Bottom panel, 1 μ m of recombinant GST ‐ PARP 10cd was automodified and treated as indicated in top panel. (D) The products of indicated enzymatic reactions were assayed by TLC.

Article Snippet: The 6xHis‐ hs PARP1 (wild‐type) was expressed and purified from E. coli , attached to MagneHis beads (Promega Corporation, Madison, WI, USA), and PARylated as described previously with the following changes: PARP1 (final concentration 1 μ m ) was autoPARylated in the presence of 1 m m β‐NAD + for 30 min at 37 °C.

Techniques: Recombinant, Incubation, Purification, SDS Page, Autoradiography, Mutagenesis

Journal: The Febs Journal

Article Title: ENPP 1 processes protein ADP ‐ribosylation in vitro

doi: 10.1111/febs.13811

Figure Lengend Snippet: ENPP 1 hydrolyses poly( ADP ‐ribose) to pR, a molecular tag detectable by LC ‐ MS / MS . (A) PARP 1 carries a 212.01 Da shift representative of pR on E168. (B) PARP 1 carries pR on E169, as shown here clearly distinguishable from the pR‐E168 peptide form. Both peptides detected following digestion of PAR by ENPP 2‐1‐T in a 10× enzyme:PARP ratio.

Article Snippet: The 6xHis‐ hs PARP1 (wild‐type) was expressed and purified from E. coli , attached to MagneHis beads (Promega Corporation, Madison, WI, USA), and PARylated as described previously with the following changes: PARP1 (final concentration 1 μ m ) was autoPARylated in the presence of 1 m m β‐NAD + for 30 min at 37 °C.

Techniques: Liquid Chromatography with Mass Spectroscopy

Journal: Molecular Cancer Therapeutics

Article Title: Nifuroxazide Activates the Parthanatos to Overcome TMPRSS2:ERG Fusion-Positive Prostate Cancer

doi: 10.1158/1535-7163.MCT-22-0159

Figure Lengend Snippet: NFZ activates parthanatos by blocking the interaction of ERG and PARP1. A, The combined effect of OLP and NFZ on VCaP cells. VCaP cells were treated with 5 μmol/L NFZ or 5 μmol/L NFZ combined with 0.5, 1.0, or 20 μmol/L OLP. The results in the histogram are shown as mean ± SD ( n = 3 independent experiments; ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001), compared with the DMSO group by Dunnett multiple comparisons test. B, Co-immunoprecipitation of ERG and PARP1 from ERG-overexpressing cells (DU145-ERG). Representative Western blotting is shown on top, and a quantitative histogram is shown beneath. Histogram results are shown as mean ± SD ( n = 5 independent experiments; ns, not significant; *, P < 0.05; **, P < 0.01), compared with the DMSO group by Dunnett's multiple comparisons test. C, Illustration of parthanatos activated by NFZ. NFZ reduces the interaction of ERG and PARP1, so that overactive PARP1 activates the parthanatos pathway. D, RT-qPCR for AIF mRNA on VCaP cells. RQ, relative quantification. Abscissa indicates 2 or 5 μmol/L NFZ treatment for 12 or 24 hours. E, AIF Western blot. F, γ-H2A.X Western blotting. Representative Western blotting is shown on top, and a quantitative histogram is shown beneath it for the diagrams in E and F. VCaP cells were treated with DMSO, or 2, or 5 μmol/L NFZ, or NFZ combined with 10 μmol/L OLP for different times. Intensity was measured using Image lab 5.2 software and normalized to total protein. Relative expression is shown using the first sample as a reference. Uncropped blots are shown in Supplementary Figs. S6, 8S and 9S. Histogram results are shown as mean ± SD ( n = 3 independent experiments; ns, not significant; *, P < 0.05;**, P < 0.01; ***, P < 0.001; ****, P < 0.0001), compared with the DMSO group by Dunnett multiple comparisons test. G, IC 50 values for ERG-overexpressing cells. The DU145 vector group is the negative control group. The DU145-ERG group is the ERG-overexpressing group, which was treated with 0.1, 0.5, 1, 5, 10, and 30 μmol/L NFZ, or a mixture of the NFZ and 10 μmol/L OLP. IC 50 values were obtained using GraphPad Prism 8.0. OLP, olaparib.

Article Snippet: The cells were incubated overnight at 4°C with primary antibodies against cleaved PARP1 (Beyotime, AF1567), AIF (Solarbio, K000163M), or phospho-histone H2A.X (Beyotime, AF1201).

Techniques: Blocking Assay, Immunoprecipitation, Western Blot, Quantitative RT-PCR, Quantitative Proteomics, Software, Expressing, Plasmid Preparation, Negative Control

Journal: Molecular Cancer Therapeutics

Article Title: Nifuroxazide Activates the Parthanatos to Overcome TMPRSS2:ERG Fusion-Positive Prostate Cancer

doi: 10.1158/1535-7163.MCT-22-0159

Figure Lengend Snippet: NFZ induces apoptosis and necrosis. A, Western blotting for cleaved PARP1. Representative Western blot is shown on top and the quantitation is shown beneath. VCaP cells were treated with DMSO, or 2, or 5 μmol/L NFZ, or NFZ combined with 10 μmol/L OLP for different times. Band intensity was measured by Image Lab 5.2 software and normalized to total protein. Relative levels are expressed using the first sample as a reference. Uncropped blots are shown in Supplementary Fig. S10. Results in the histogram are shown as mean ± SD ( n = 3 independent experiments; ns, not significant; *, P < 0.05;**, P < 0.01; ***, P < 0.001; ****, P < 0.0001), compared with the DMSO group by Dunnett multiple comparisons test. B, Confocal laser imaging for cleaved PARP1 and AIF. VCaP cells were treated with 2 μmol/L NFZ for 30 minutes. Immunofluorescence of cleaved PARP1 (green), AIF (red) and nuclei (DAPI, blue). Scale bars, 5 μm. C, Confocal laser imaging for γ-H2A.X. VCaP cells were treated with 2 μmol/L NFZ for 12 hours. Immunofluorescence of γ-H2A.X (green) and nuclei (DAPI, blue). Scale bars, 5 μm. D, Flow cytometry for cell death. Apoptotic cells and dead cells are stained by Hoechst to different degrees. VCaP cells were treated with 5 μmol/L NFZ, 10 μmol/L NFZ, or 20 μmol/L NFZ for 48 hours or 72 hours. Q1 and Q2 gate are the necrotic cells; Q3 and Q4 represent the normal cells and apoptotic cells, respectively. The results show the proportion of dead cells to the total number with at least 10,000 cells counted. DMSO was used as a control. OLP, olaparib.

Article Snippet: The cells were incubated overnight at 4°C with primary antibodies against cleaved PARP1 (Beyotime, AF1567), AIF (Solarbio, K000163M), or phospho-histone H2A.X (Beyotime, AF1201).

Techniques: Western Blot, Quantitation Assay, Software, Imaging, Immunofluorescence, Flow Cytometry, Staining, Control

Journal: Frontiers in Pharmacology

Article Title: Alkaloid from Alstonia yunnanensis diels root against gastrointestinal cancer: Acetoxytabernosine inhibits apoptosis in hepatocellular carcinoma cells

doi: 10.3389/fphar.2022.1085309

Figure Lengend Snippet: Effects of AC on the apoptosis-related signaling pathway proteins. (A , B) Effect of AC on mitochondrial membrane potential in SMMC7721. (C , D) Effect of AC on mitochondrial membrane potential in BEL-7402. (E , F) Western blot detection of Parp-1, Cleaved-parp-1, Caspase9, Cleaved-caspase9, Caspase3, and Active-caspase3 expression and statistical results in SMMC7721. (G , H) Western blot detection of Parp-1, Cleaved-parp-1, Caspase9, Cleaved-caspase9, Caspase3, and Active-caspase3 expression and statistical results in BEL-7402. * p < 0.05; ** p < 0.01; *** p < 0.001 (vs . 0 group).

Article Snippet: AnnexinV/PI cell double staining apoptosis detection kit, TUNEL (ALEX647) apoptosis detection kit, cell cycle detection kit, and DAPI anti-fluorescence quenching mounting tablets were purchased from Shanghai Yisheng Biotechnology Co. Ltd. Sulfonyl Rhodamine B (SRB) was purchased from Shanghai Aladdin Reagent Co. Ltd. Tris (Tris) was purchased from Shanghai Shenggong Biological Engineering Co. Ltd. Trichloroacetic acid (TCA) was purchased from Shanghai Saen Chemical Technology Co. Ltd. Anti-Caspase3, anti-Caspase9, anti-Parp-1, anti-Cleaved-parp-1 were purchased from Shanghai Shenggong BBILIFE.

Techniques: Western Blot, Expressing

Journal: PLoS ONE

Article Title: M30 Antagonizes Indoleamine 2,3-Dioxygenase Activation and Neurodegeneration Induced by Corticosterone in the Hippocampus

doi: 10.1371/journal.pone.0166966

Figure Lengend Snippet: Levels of protein expression of (A) Bcl-2, (B) Cleaved Caspase 3 and (C) Cleaved PARP-1 in the hippocampus of the control, CORT-treated (CORT), M30-treated (M30), CORT and M30 co-treated (CORT+M30) or vehicle groups are summarized. β-actin was an internal control. Data from each group were expressed as mean ± SEM (n = 8). Statistical comparisons between groups were performed using the One way Anova followed by Tukey post hoc test to detect differences in all groups. For Bcl-2, *p < 0.01 when compared with Control, # p < 0.01 when compared with M30, $ p < 0.01 when compared with CORT + M30 groups, ! p < 0.01 when compared with Vehicle. For cleaved caspase 3 and cleaved PARP-1, *p < 0.001 when compared with Control, # p < 0.001 when compared with M30, $ p < 0.001 when compared with CORT + M30 groups, ! p < 0.001 when compared with Vehicle.

Article Snippet: Primary antibodies of SOD-2 (rabbit polyclonal, 1:1000), GPx-1 (goat polyclonal, 1:500), NFκB p65 (rabbit polyclonal, 1:250) and p50 (mouse monoclonal, 1:250), IκBα (mouse monoclonal, 1:500), TNF α (goat polyclonal, 1:80), IL-1β (rabbit polyclonal 1:100), IL-6 (goat polyclonal, 1:1000) and COX-2 (goat polyclonal, 1:100) were purchased from Santa Cruz Biotechnology, CA, USA; Synapsin 1 (rabbit polyclonal, 1:500) and Synaptophysin (rabbit polyclonal, 1:2000) were purchased from Novus Biologicals, USA; PSD95 (rabbit polyclonal 1:500), Cleaved Caspase 3 (rabbit polyclonal 1:500) was purchased from Cell Signaling Technology; Cleaved PARP-1 (rabbit polyclonal, 1:2000) was purchased from Bioworld Technology; IDO-1 (rabbit polyclonal, 1:250) was purchased from antibodies-online (ABIN1714836).

Techniques: Expressing

Journal: British Journal of Cancer

Article Title: Targeting ADAM-17 with an inhibitory monoclonal antibody has antitumour effects in triple-negative breast cancer cells

doi: 10.1038/bjc.2015.163

Figure Lengend Snippet: Effects of D1(A12) on cell death of HCC1143 breast cancer cells. Cells were treated for 48 h with IgG control, D1(A12) (200 n M ) or Ab17 (0.5 μ g ml −1 ). ( A ) Cell lysates were blotted with a PARP antibody and the relative amount of full-length and cleaved PARP was measured using β -actin as a loading control. A representative blot is shown with the mean densitometric analysis of four experiments. * P <0.05, ** P <0.001 compared with IgG control. Oligonucleosomes were measured by ELISA from ( B ) cell lysates and ( C ) cell culture supernatants from 3D scaffolds.

Article Snippet: Apoptosis was also evaluated by immunoblotting for cleaved PARP1 (Fisher Scientific, Dublin, Ireland).

Techniques: Control, Enzyme-linked Immunosorbent Assay, Cell Culture