Journal: Dose-Response

Article Title: Gentiana lutea Root Extract Attenuates Radiation-Induced Damage in Human PBMCs in vitro

doi: 10.1177/15593258261435484

Figure Lengend Snippet: Relative fold change of genes expression involved in DNA repair processes: Ogg1, Parp1 , and Xrcc1 in PBMCs pretreated with 0.25, 0.5, 1 and 2 mg/mL of Gentiana lutea root extract and exposed to 0.5 and 2 Gy irradiation dose

Article Snippet: Expression levels of the target genes were measured by quantitative Real-time PCR on Applied Biosystems Real-Time 7500 system (Applied Biosystems, Inc., Foster City, CA) using TaqMan® gene expression assays: Parp1 (Hs00242302_m1), Ogg1 (Hs00213454_m1), Xrcc1 (Hs00959834_m1).

Techniques: Expressing, Irradiation

Journal: Nucleic Acids Research

Article Title: PARP10 promotes cellular proliferation and tumorigenesis by alleviating replication stress

doi: 10.1093/nar/gky658

Figure Lengend Snippet: Loss of PARP10 impairs proliferation of HeLa cells. ( A ) Western blot showing loss of PARP10 expression in HeLa cells with CRISPR/Cas9-mediated PARP10 knockout. ( B ) PARP10-knockout HeLa cells show reduced proliferation rates. The average of three experiments with error bars representing standard deviations is shown. The asterisk indicates statistical significance (using the two-tailed equal variance TTEST). ( C ) Representative clonogenic assay showing reduced proliferation of PARP10-knockout HeLa cells. ( D ) Representative PI flow cytometry profile showing an altered cell-cycle distribution in PARP10-knockout HeLa cells. ( E ) Quantification of cell-cycle distribution in control and PARP10-knockout HeLa cells. The average of four experiments, with error bars as standard deviations, is shown. Statistical significance was calculated using the two-tailed equal variance TTEST. ( F ) Western blot showing the re-expression of PARP10, with a Myc-tag, in PARP10-knockout HeLa cells. ( G ) Exogenous PARP10 expression rescues the proliferation defect of PARP10-deleted HeLa cells. The average of four experiments with error bars representing standard deviations is shown. The asterisk indicates statistical difference between the PARP10 KO and PARP10 KO + Myc-PARP10 samples.

Article Snippet: Antibodies used for western blot were: PARP10 (Novus NB100–2157), GAPDH (Santa Cruz Biotechnology sc-47724), Polη (Cell Signaling Technology 13848), PCNA (Cell Signaling Technology 2586), Ubiquityl-PCNA Lys164 (Cell Signaling Technology 13439).

Techniques: Western Blot, Expressing, CRISPR, Knock-Out, Two Tailed Test, Clonogenic Assay, Flow Cytometry

Journal: Nucleic Acids Research

Article Title: PARP10 promotes cellular proliferation and tumorigenesis by alleviating replication stress

doi: 10.1093/nar/gky658

Figure Lengend Snippet: Loss of PARP10 results in sensitivity to replication stress. ( A ) Annexin V apoptosis experiment showing increased apoptosis in PARP10-knockout cells following HU treatment. Cells were treated with 1 mM HU for 24 h. Data are shown as normalized to the control (no drug treatment) condition for each cell line. The mean of six experiments with error bars as standard deviations is shown. ( B ) Clonogenic assay showing that PARP10-knockout cells are sensitive to HU, and re-expression of PARP10 corrects this sensitivity. For each cell line, the ratios of HU-treated to non-treated are presented. The mean and standard deviation are shown. ( C ) Quantification of the proportion of cells in G1 at the indicated time points after release from HU (1 mM for 24 h). The mean and standard deviation are shown. Representative flow cytometry histograms are shown in . ( D ) Schematic representation of the DNA fiber combing assay condition, including a representative micrograph. ( E ) DNA fiber combing assay showing reduced replication fork progression in PARP10-knockout cells upon HU exposure. Shown is the quantification of the IdU tract length, with the median values marked. Representative micrographs for each condition are presented in .

Article Snippet: Antibodies used for western blot were: PARP10 (Novus NB100–2157), GAPDH (Santa Cruz Biotechnology sc-47724), Polη (Cell Signaling Technology 13848), PCNA (Cell Signaling Technology 2586), Ubiquityl-PCNA Lys164 (Cell Signaling Technology 13439).

Techniques: Knock-Out, Clonogenic Assay, Expressing, Standard Deviation, Flow Cytometry

Journal: Nucleic Acids Research

Article Title: PARP10 promotes cellular proliferation and tumorigenesis by alleviating replication stress

doi: 10.1093/nar/gky658

Figure Lengend Snippet: Overexpression of PARP10 promotes proliferation of non-transformed RPE-1 cells. ( A ) Schematic representation of PARP10 domain organization. The black line underneath shows the length of the PARP10-ΔPARP variant (spanning residues 1–834) which lacks the PCNA-interacting PIP motif and the catalytic PARP domain. RRM: RNA recognition motif; NES: nuclear localization signal; UIM: ubiquitin interacting motifs; PIP: PCNA-interacting motif; PARP: catalytic ADP-ribosyltransferase domain. ( B ) Western blot showing the overexpression of Myc-tagged PARP10 wild-type and ΔPARP in RPE-1 cells. ( C – E ) Overexpression of wild-type, but not PARP-deleted PARP10, promotes proliferation of RPE-1 cells. (C) Representative clonogenic assay. (D) Quantification of cell number from clonogenic assays using CellTiterGlo reagent. The mean and standard deviation are shown. (E) Quantification of EdU-incorporating cells. EdU was added to the media for 45 min prior to harvesting. The mean with standard deviation is shown. Representative flow cytometry plots are shown in . ( F ) DNA fiber combing assay showing increased replication tracts in PARP10-overexpressing RPE-1 cells under normal (no drug treatment) conditions. Shown is the quantification of the IdU tract length, with the median values marked.

Article Snippet: Antibodies used for western blot were: PARP10 (Novus NB100–2157), GAPDH (Santa Cruz Biotechnology sc-47724), Polη (Cell Signaling Technology 13848), PCNA (Cell Signaling Technology 2586), Ubiquityl-PCNA Lys164 (Cell Signaling Technology 13439).

Techniques: Over Expression, Transformation Assay, Variant Assay, Western Blot, Clonogenic Assay, Standard Deviation, Flow Cytometry

Journal: Nucleic Acids Research

Article Title: PARP10 promotes cellular proliferation and tumorigenesis by alleviating replication stress

doi: 10.1093/nar/gky658

Figure Lengend Snippet: Overexpression of PARP10 in RPE-1 suppresses replication stress and promotes engagement of mutagenic TLS polymerase Polη. ( A ) Clonogenic assay showing that PARP10-overexpressing RPE-1 cells are resistant to HU. For each cell line, the ratios of HU-treated to non-treated are presented. The mean and standard deviation are shown. ( B ) DNA fiber combing assay showing increased replication tracts in PARP10-overexpressing RPE-1 cells under HU treatment. Shown is the quantification of the IdU tract length, with the median values marked. ( C ) Chromatin fractionation experiments showing increased chromatin recruitment of TLS polymerase Polη in PARP10-overexpressing RPE-1 cells following HU treatment (2 mM for 24 h). A repeat experiment, as well as quantification of band intensities are provided in . ( D ) Mutation load in control and PARP10-overexpressing RPE-1 cells calculated from RNA-seq data. Three independent clones each were grown for 30 generations in the presence of doxycycline to induce PARP10 expression. The number of point mutations from loci with at least 10 reads was calculated, and normalized against genome coverage. The mean values are shown ( P = 0.55).

Article Snippet: Antibodies used for western blot were: PARP10 (Novus NB100–2157), GAPDH (Santa Cruz Biotechnology sc-47724), Polη (Cell Signaling Technology 13848), PCNA (Cell Signaling Technology 2586), Ubiquityl-PCNA Lys164 (Cell Signaling Technology 13439).

Techniques: Over Expression, Clonogenic Assay, Standard Deviation, Fractionation, Mutagenesis, RNA Sequencing Assay, Clone Assay, Expressing

Journal: Nucleic Acids Research

Article Title: PARP10 promotes cellular proliferation and tumorigenesis by alleviating replication stress

doi: 10.1093/nar/gky658

Figure Lengend Snippet: PARP10 promotes tumor growth in vivo . ( A and B ) PARP10 deletion reduces tumor formation by HeLa cells. (A) Quantification of tumor size, 28 days after subcutaneous injection. A total of eight mice were used for each condition. The mean and standard deviation are shown. (B) Representative images of tumor formation by HeLa control and PARP10-knocokout cells at day 28. ( C ) Exogenous re-expression of Myc-tagged PARP10 restores tumor formation ability. Quantification of tumor size, 28 days after subcutaneous injection is shown. For each condition, five mice were used, which were administered doxycycline in their drinking water to induce exogenous Myc-PARP10 expression. The mean and standard deviation are shown. ( D and E ) PARP10 overexpression promotes tumor formation by non-transformed RPE-1 cells. (D) Quantification of tumor size, 28 days after subcutaneous injection. For each condition, seven mice were used, which were administered doxycycline in their drinking water to induce exogenous Myc-PARP10 expression. The mean and standard deviation are shown. (E) Representative images of tumor formation by RPE-1 cells at day 28. ( F ) Model showing the involvement of PARP10 in carcinogenesis. PARP10 overexpression during transformation confers protection against replication stress by increasing TLS. Targeting PARP10 may reduce proliferation of cancer cells.

Article Snippet: Antibodies used for western blot were: PARP10 (Novus NB100–2157), GAPDH (Santa Cruz Biotechnology sc-47724), Polη (Cell Signaling Technology 13848), PCNA (Cell Signaling Technology 2586), Ubiquityl-PCNA Lys164 (Cell Signaling Technology 13439).

Techniques: In Vivo, Injection, Standard Deviation, Expressing, Over Expression, Transformation Assay

Journal: Annals of Hematology

Article Title: Inhibition of CDC20 suppresses the development and progression of mantle cell lymphoma through PI3K/AKT pathway

doi: 10.1007/s00277-026-06926-0

Figure Lengend Snippet: Apcin demonstrated anti-tumor efficacy and safety in the Z138 xenograft model. ( A ) Tumor growth curves of the control group and the apcin group. ( B ) Comparison of tumor weight between the two groups. ( C ) Comparison of spleen weight between the two groups. ( D and E ) Representative photographs of harvested tumors ( D ) and spleens ( E ) from both groups. ( F ) Body weight monitoring of mice in the control group and the apcin group. ( G ) Representative HE images (×200 magnification; scale bar, 100 μm) of heart, liver, and kidney tissues of mice in both groups. ( H ) Comparison of relevant parameters in blood biochemistry of mice between the two groups. ( I ) Comparison of relevant parameters in blood routine of mice between the two groups. ( J ) Representative IHC images of CDC20, cleaved PARP and Ki67 staining of tumor tissues in the control group and the apcin group (×200 magnification; scale bar, 100 μm). ( K ) Quantification analysis of CDC20, cleaved PARP and Ki67 expression in the control group and the apcin group calculated by MOD values. ( L ) WB validation of CDC20 and cleaved PARP expression in both groups. ** P < 0.01, *** P < 0.001, ns meant no significance

Article Snippet: Subsequently, sections were incubated overnight at 4 °C with primary antibodies: CDC20 for patients (1:500, 10252-1-AP, Proteintech, China) and mouse tumors (1:200, 10252-1-AP, Proteintech, China), and cleaved PARP (1:200, #94885, CST, USA) and Ki-67 (1:400, #12202, CST, USA) for mouse tumors.

Techniques: Control, Comparison, Staining, Expressing, Biomarker Discovery

Journal: Molecular Cancer Research

Article Title: Constitutive Activation of Caspase-3 and Poly ADP Ribose Polymerase Cleavage in Fanconi Anemia Cells

doi: 10.1158/1541-7786.mcr-09-0373

Figure Lengend Snippet: FIGURE 1. PARP cleavage and upregulation in FA cells. A. Immunoblot showing PARP cleavage in FANCD2-deficient (PD20), FANCD2-proficient (PD20cor), or nonmonoubiquitinable mutant (K561R) cells. B. Immunoblot showing PARP processing in PD20 and PD20-corrected cells treated (+) or untreated (−) with PARP inhibitor 3AB. C. PARP cleavage is increased in primary fibroblasts transfected with FANCD2 siRNA for the corresponding time intervals. Arrows, mobility shift representing PARP phosphorylation. Note the reduction of PARP signal in the sample treated with λ-phosphatase or in the siRNA FANCD2–depleted cells pretreated with PKC inhibitor. D. Immunoblot of the above FANCD2 knockdown samples immunoprecipitated with PARP antibody followed by probing with phosphor-PARP or total PARP antibodies. E. Immunoblots showing PARP cleavage in different FA subgroups treated or untreated with the PARP inhibitor, 3AB.

Article Snippet: In addition to the antibodies used for Western blotting, cleaved PARP (Asp214) antibody (human specific) #9541 (Cell Signaling, Inc.) has been used.

Techniques: Western Blot, Mutagenesis, Transfection, Mobility Shift, Phospho-proteomics, Knockdown, Immunoprecipitation

Journal: bioRxiv

Article Title: Non-DNA-damaging DNA-PK activation improving hearing and prolonging life due to NAD + and SIRT upregulation

doi: 10.1101/2025.04.18.649305

Figure Lengend Snippet: a, Separation of MA-5 R-enantiomer and S-enantiomer from MA-5 racemic body by the chiral column (upper). The chemical inversion/conversion of R -and S- enantiomer of MA-5 were not observed in a rhesus monkey administered each enantiomer (lower). b , Intracellular ATP measurement of HEI-OC1 cells with various concentration of MA-5, S-enantiomer and R-enantiomer at 0, 1, 3,10, 30 and 100nM for 6hr. Data were mean ± SE.* p <0.05, ** p <0.01 vs. DMSO control; statistics were calculated by One way anova Durnett’s multiple comparison test. c , The cell-protective effect of MA-5 S-enantiomer and R-enantiomer against cytotoxicity of BSO in skin fibroblasts from a MELAS patient. MELAS patient skin fibroblasts were cultured with BSO at 500μM through various concentrations of MA-5 S-enantiomer (left) or R-enantiomer (right) at 10, 30, 100, 300nM, 1 μM, 3μM 10 μM and 100 μM for 72hrs. Cell viability and cytotoxicity were measured by WST-8 assay. Data were mean ± SE.* p <0.05, ** p <0.01, *** p <0.001 vs 0.1% DMSO control; statistics were calculated by One way anova Durnett’s multiple comparison test. d, Intracellular NAD + measurement of HEI-OC1 cells. HEI-OC1 cells were treated with 0.1 % DMSO or MA-5 racemate, MA-5 S-enantiomer and R- enantiomer at 3,10, 30 and 100nM for 6hr. Data were mean ± SE. * p <0.05, ** p <0.01 vs. DMSO control; statistics were calculated by One way anova Durnett’s multiple comparison test. e , Intracellular NAD + measurement of human iPS-induced inner ear cells. Human iPS-induced inner ear cells were treated with 0.1 % DMSO or MA-5 racemate, MA-5 S-enantiomer and R-enantiomer at 3,10, 30 and 100nM for 6hr. Data were mean ± SE. * p <0.05, ** p <0.01 vs. DMSO control; statistics were calculated by One way anova Durnett’s multiple comparison test. f, The effects of S-enantiomer on SIRT1 enzymatic activity. The effects of S- enantiomer on SIRT1 deacetylase activity in vitro at 10,30 and 100 μM (left). The effects of S-enantiomer on SIRT1 deacetylase activity in cell nuclear fraction extract from HEI-OC1 cells treated with S-enantiomer at 10 and 30 μM for 3hr (right). Data were mean ± SE.* p <0.05, ** p <0.01 vs. DMSO control; statistics were calculated by One way anova Durnett’s multiple comparison test. g, The acetylation of p53 (K305) was reduced by MA-5 S enantiomer. The effects of S-enantiomer on the acetylation of p53 (K305) in whole cell extraction from HEI-OC1 cells treated with MA-5 racemate and S-enantiomer at 10 and 30 μM for 24hr. h , The cellular localization of PARylation in HEI-OC1 cells treated with 0.1 % DMSO or MA-5 at 10μM for 24hr. (left). Poly-ADP ribosylation of PARP protein in HEI-OC1 cells treated with 0.1 % DMSO, MA-5 racemate, S-enantiomer and R-enantiomer at 30 100μM for 24hr. i , Immunocyto-staining of H2AX phosphorylated (γH2AX) of HEI-OC1 cells treated with 0.1 % DMSO, doxiorubicin (Dox) at 0.3μM, MA-5, S-enantiomer and R-enantiomer at 30 μM for 24hr. Representative (Upper). Florescence intensities of γH2AX immunocytostaining were measured (Lowere). Data were mean ± SE. p <0.05, ***: p <0.0001 vs. control. # p <0.05, #### p <0.0001 vs. Dox 0.3μM; statistics were calculated by One way anova Durnett’s multiple comparison test. j , Immunocytostaining of H2AX phosphorylated (γH2AX) of HEI-OC1 cells treated with 0.1 % DMSO, camptothecin (Capt) at 1μM, MA-5, S-enantiomer and R-enantiomer at 30 μM for 24hr. Representative (Upper). Florescence intensities of γH2AX immunocytostaining were measured (Lowere). Data were mean ± SE. p <0.05, ***: p <0.0001 vs. control. # p <0.05, #### p <0.0001 vs. Capt 1μM; statistics were calculated by One way anova Durnett’s multiple comparison test. k , The NAMPT enzymatic activity in vitro treated with MA-5, S-enantiomer and R- enantiomer at 3,10, 30 and 100nM for 1hr. Data were mean ± SE. * p <0.05, ** p <0.01 vs. DMSO control; statistics were calculated by One way anova Durnett’s multiple comparison test. l, The NAMPT enzymatic activity in vitro treated with S-enantiomer at 10 and 100μM with or without selective NAMPT inhibitor FK-866 at 10nM ( left ). Data were mean ± SE.* p <0.05, ** p <0.01 vs. DMSO control; statistics were calculated by One way anova Durnett’s multiple comparison test. The Inhibitory curve on NAMPT activity with various concentration of FK-866 at at 10, 20, 30, 40 and 50nM ( right ). The Inhibitory curve on NAMPT activity with FK-866 was shifted by addition of MA-5 S-enantiomer at 100 μM ( right ). * p <0.05, ** p <0.01 vs. FK-866 alone inhibitory curve; s statistics were calculated by unpaired two-side t-test. m , The binding between NAMNPT and MA-5, MA-5 S-enantiomer and R-enantiomer and FK-866 were quantitatively analyzed using Biolayer Interferometry (BLI). n , In silico study of Mitochonic acid 5 binding to NAMPT. (a) Possible binding sites on NAMPT of MA-5 S-enantiomer and R-enantiomer. (b) The binding interaction scores of MA-5 S-enantiomer and R-enantiomer at sites ( i ) and ( ii ). (c) The binding conformations of MA-5 S-enantiomer and R-enantiomer at sites ( i ) and ( ii ). (d) the conformational dynamics of ligand binding of MA-5 S-enantiomer and R-enantiomer. (e)

Article Snippet: CYcLex, MBL-Life Science) and Universal Colorimetric PARP Assay Kit with Histone-Coated Strip Wells (Cat. # 4677-096-K, R & D systems) respectively according to manufacture instruction.

Techniques: Concentration Assay, Control, Comparison, Cell Culture, Activity Assay, Histone Deacetylase Assay, In Vitro, Extraction, Staining, Binding Assay, In Silico, Ligand Binding Assay