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: Antibodies from the following companies were used for Western blot analysis: SIRT1 (1:1000 dilution, #9475, Cell Signaling), SIRT2 (1:1000 dilution, #12650, Cell Signaling), SIRT3 (1:1000 dilution, #5490, Cell Signaling), SIRT4 (1:1000 dilution, #69786, Cell Signaling), SIRT5(1:1000 dilution, #8782, Cell Signaling), SIRT6 (1:1000 dilution, #12486, Cell Signaling), SIRT7(1:1000 dilution, sc-365344,Santa Cruz), IMMT/Mitofilin (1:1000 dilution, 10179-1-AP, ProteinTech), p53 (1:1000 dilution, #2524, Cell Signaling), acertyl p53(K305) (1:1000 dilution, ab109396, abcam), Phospho-p53 (Ser15) (1:1000 dilution, #9284, Cell Signaling), TRIM28(KAP1)(1:10000 dilution, ab109287, abcam), 4H11 pTRIM28 (Ser 473) mouse mAb(1:1000 dilution, Gift from Dr .Shunsuke Chikuma), pTRIM28(KAP1) (Ser 824) (1:1000 dilution, ab133440, abcam), ATM (1:1000 dilution, #2873, Cell Signaling), pATM(Ser 1981)(1:1000 dilution, Invitrogen #14-9046-82), DNA-PKcs (1:1000 dilution, #4602, Cell Signaling), pDNA-PKcs(Ser 2056) (1:1000 dilution, ab18192, abcam), K48-linkage Specific Polyubiquitin (1:1000 dilution, #4289, Cell Signaling), FLAG® tag epitope(DYKDDDDK tag) (1:1000 dilution, F1804, Sigma Aldrich), MYC Tag (1:1000 dilution, #2278, Cell Signaling), PARP1 (1:1000 dilution, #9542, Cell Signaling), PARP1, acetyl Lys521 (1:1000 dilution, CSB-PA890185, CUSABIO), poly(ADP-ribose) polymer (pADPr) (1:1000 dilution, sc-56198, Santa Cruz), 53BP1 (1:1000 dilution, ab175933, Abcam), Phospho-53BP1 (Ser25) (1:1000 dilution, NB100-1803, Novus Biologicals), Acetyl-Histone H3 (Lys18) (1:1000 dilution, #9675, Cell Signaling), Phospho-Chk2 (Thr68) (1:1000 dilution, E-AB-20843, Elabscience), DNA PKcs (pS2056) (1:1000 dilution, ab124918, Abcam), GAPDH (1:1000 dilution, sc-32233, Santa Cruz), β-actin (1:500 dilution, sc-47778, Santa Cruz).

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

Journal: iScience

Article Title: PBX3 regulates mast cell parthanatos via TOP2A mediated DNA damage in allergic rhinitis

doi: 10.1016/j.isci.2026.115426

Figure Lengend Snippet: Regulatory role of TOP2A in mast cell apoptosis, parthanatos pathway, and inflammatory response In vitro experimental groups: CON, model, Model+OE-NC, Model+OE-TOP2A, Model+si-NC, Model+si-TOP2A. (A and B) RT-qPCR and western blot analyses were conducted to assess the mRNA and protein expression levels of TOP2A and PARP-1 in mast cells. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. the CON or Model+OE-NC or Model+si-NC group. (C) TUNEL staining was performed to evaluate and visualize cell apoptosis. ∗ p < 0.05, ∗∗ p < 0.01 vs. the CON or Model+OE-NC or Model+si-NC group; scale bars, 50 μm. (D) Cell proliferation activity in each group was measured using the CCK-8 assay. ∗∗ p < 0.01 vs. the CON or Model+OE-NC or Model+si-NC group. (E and F) MMP was assessed using the JC-1 assay to evaluate mitochondrial function. ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. the CON or Model+OE-NC or Model+si-NC group. (G) Western blotting was used to examine the protein levels of mitochondrial AIF (Mito-AIF), cytoplasmic AIF (Cyto-AIF), and nuclear AIF (Nucleo-AIF), along with visual analysis. ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. the CON or Model+OE-NC or Model+si-NC group. (H and I) ELISA was employed to determine the concentrations of β-hexosaminidase, Histamine, IL-4, IL-5, and IFN-γ. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. the CON or Model+OE-NC or Model+si-NC group. Data are represented as mean ± SEM ( n = 3 per group) from independent biological replicates. One-way ANOVA with Tukey’s post hoc correction for (A–D) and (F–I).

Article Snippet: Cells were then incubated overnight at 4°C with a primary antibody against PARP-1 (PB9309, BOSTER, Pleasanton, CA, USA, 1:100).

Techniques: In Vitro, Quantitative RT-PCR, Western Blot, Expressing, TUNEL Assay, Staining, Activity Assay, CCK-8 Assay, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: PBX3 regulates mast cell parthanatos via TOP2A mediated DNA damage in allergic rhinitis

doi: 10.1016/j.isci.2026.115426

Figure Lengend Snippet: Effects of TOP2A on DNA damage, parthanatos regulation, and degranulation in mast cells Experimental groups: Model+OE-NC, Model+OE-TOP2A, Model+si-NC, Model+si-TOP2A. (A) Western blotting was employed to determine the protein expression levels of γ-H2AX. ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. the Model+OE-NC or Model+si-NC group. (B and C) IF staining was performed to assess the expression of the DNA damage marker γ-H2AX in mast cells. ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. the Model+OE-NC or Model+si-NC group; scale bar = 50 μm. (D) Cellular senescence was evaluated using an SA-β-gal staining kit. ∗ p < 0.05, ∗∗ p < 0.01 vs. the Model+OE-NC or Model+si-NC group; scale bars, 50 μm. Experimental groups: Model+OE-NC, Model+OE-TOP2A, Model+OE-TOP2A + DOX. (E and F) RT-qPCR and western blot analyses were conducted to detect the gene and protein expression levels of TOP2A and PARP-1 under different treatment conditions. ∗∗∗ p < 0.001 vs. the Model+OE-NC or Model+OE-TOP2A group; ns, not significant ( p ≥ 0.05). (G) Western blotting was employed to determine the protein expression levels of γ-H2AX. ∗∗∗ p < 0.001 vs. the Model+OE-NC or Model+OE-TOP2A group. (H) IF staining was used to evaluate γ-H2AX expression as a marker of DNA damage. ∗ p < 0.05, ∗∗∗ p < 0.001 vs. the Model+OE-NC or Model+OE-TOP2A group; scale bars, 50 μm. (I) SA-β-gal staining was used to assess cellular senescence. ∗ p < 0.05, ∗∗ p < 0.01 vs. the Model+OE-NC or Model+OE-TOP2A group; scale bars, 50 μm. (J) Western blot analysis was used to determine the protein expression levels of Mito-AIF, Cyto-AIF, and Nucleo-AIF. ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. the Model+OE-NC or Model+OE-TOP2A group. (K and L) ELISA assays were carried out to measure the levels of β-hexosaminidase, Histamine, IL-4, IL-5, and IFN-γ. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. the Model+OE-NC or Model+OE-TOP2A group. Data are represented as mean ± SEM ( n = 3 per group) from independent biological replicates. one-way ANOVA with Tukey’s post hoc correction for (A and C–L).

Article Snippet: Cells were then incubated overnight at 4°C with a primary antibody against PARP-1 (PB9309, BOSTER, Pleasanton, CA, USA, 1:100).

Techniques: Western Blot, Expressing, Staining, Marker, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: PBX3 regulates mast cell parthanatos via TOP2A mediated DNA damage in allergic rhinitis

doi: 10.1016/j.isci.2026.115426

Figure Lengend Snippet: Effects of PBX3 silencing on mast cell function and DNA damage response Experimental groups: Model+si-NC, Model+ si-PBX3. (A) RT-qPCR and western blot analyses were conducted to evaluate the mRNA expression levels of PBX3 and TOP2A. ∗∗∗ p < 0.001 vs. the Model+si-NC group. (B–D) Western blotting and IF staining were used to assess the expression of the DNA damage marker γ-H2AX. ∗ p < 0.05, ∗∗∗ p < 0.001 vs. the Model+si-NC group; scale bars, 50 μm. (E) SA-β-gal staining was performed to detect cellular senescence. ∗∗ p < 0.01 vs. the Model+si-NC group; scale bars, 50 μm. (F–H) RT-qPCR, western blotting, and IF staining were employed to examine PARP-1 expression in mast cells. ∗∗∗ p < 0.001 vs. the Model+si-NC group; scale bars, 50 μm. (I) Western blot analysis was used to detect the protein levels of Mito-AIF, Cyto-AIF, and Nucleo-AIF. ∗∗∗ p < 0.001 vs. the Model+si-NC group. (J and K) ELISA assays were conducted to quantify the levels of β-hexosaminidase, histamine, IL-4, IL-5, and IFN-γ in mast cells. ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. the Model+si-NC group. Data are represented as mean ± SEM ( n = 3 per group) from independent biological replicates. Two-tailed unpaired Student’s t tests for (A, B, D–G, and I–K).

Article Snippet: Cells were then incubated overnight at 4°C with a primary antibody against PARP-1 (PB9309, BOSTER, Pleasanton, CA, USA, 1:100).

Techniques: Cell Function Assay, Quantitative RT-PCR, Western Blot, Expressing, Staining, Marker, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: iScience

Article Title: PBX3 regulates mast cell parthanatos via TOP2A mediated DNA damage in allergic rhinitis

doi: 10.1016/j.isci.2026.115426

Figure Lengend Snippet: PBX3 drives DNA damage through TOP2A to regulate mast cell parthanatos, senescence, and degranulation Experimental groups: Model+si-NC, Model+si-PBX3, Model+si-PBX3+OE-NC, Model+si-PBX3+OE-TOP2A. (A) Western blot analysis was performed to determine protein expression levels of PBX3, TOP2A, PARP-1, and γ-H2AX. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. the Model+si-NC or Model+si-PBX3+OE-NC group; ns, not significant ( p ≥ 0.05). (B) IF staining was conducted to detect the DNA damage marker γ-H2AX. ∗∗∗ p < 0.001 vs. the Model+si-NC or Model+si-PBX3+OE-NC group; scale bars, 50 μm. (C) Cellular senescence was assessed using an SA-β-gal staining kit. ∗∗∗ p < 0.001 vs. the Model+si-NC or Model+si-PBX3+OE-NC group; scale bars, 50 μm. (D) ELISA was employed to measure the levels of degranulation markers β-hexosaminidase and histamine. ∗∗ p < 0.01, ∗∗∗ p < 0.001 vs. the Model+si-NC or Model+si-PBX3+OE-NC group. (E) Cytokine levels of IL-4, IL-5, and IFN-γ in cell supernatants were quantified by ELISA. ∗ p < 0.05, ∗∗∗ p < 0.001 vs. the Model+si-NC or Model+si-PBX3+OE-NC group. Data are represented as mean ± SEM ( n = 3 per group) from independent biological replicates. One-way ANOVA with Tukey’s post hoc correction for (A–E).

Article Snippet: Cells were then incubated overnight at 4°C with a primary antibody against PARP-1 (PB9309, BOSTER, Pleasanton, CA, USA, 1:100).

Techniques: Western Blot, Expressing, Staining, Marker, Enzyme-linked Immunosorbent Assay

Journal: Molecular Oncology

Article Title: HSP90 identified by a proteomic approach as druggable target to reverse platinum resistance in ovarian cancer

doi: 10.1002/1878-0261.12883

Figure Lengend Snippet: HSP90 pharmacological inhibition or HSP90α knockout increases the pro‐apoptotic and DNA damage effect of CDDP in Pt‐res TOV‐112D cells. (A) Apoptosis and necrosis evaluated by flow cytometry after Annexin V‐FITC and propidium iodide staining in TOV‐112D Pt‐res cl. 7 and (B) cl. 2 cells, untreated or treated for 48 h (upper panels) or 72 h (lower panels), with CDDP and/or ganetespib at IC 50 72 h doses of parental cells. (C) Western blot analysis of γH2AX, PARP1 and caspase 3 cleavage, BAX and BCL2 expression, in TOV‐112D Pt‐res pool 2 cells untreated or treated with CDDP and/or ganetespib at the doses indicated above. β‐Actin expression serves as loading control. Western blot quantification was performed by imagej software. (D) Apoptosis and necrosis evaluated by flow cytometry after Annexin V‐FITC and propidium iodide staining in TOV‐112D, TOV‐112D Pt‐res cl. 7 and in HSP90α knockout TOV‐112D Pt‐res KO#2 cells, untreated or treated for 48 or 72 h with CDDP at IC 50 72 h doses of parental cells.

Article Snippet: Primary antibodies were purchased as follows: HSP90 alpha 2G5.G3 (#SMC‐147) and HSP90 (total) 4F3.E8 (#SMC‐149) from StressMarq Biosciences (Victoria, BC, Canada); GRP75 (#3593), LMN A/C (#2032), ANXA1 (#3299), Bax (#2774), CASPASE 3 (#9662), cleaved PARP1 (Asp214; #5625), PARP1 (#9542), Bcl‐2 (#4223S) from Cell Signaling Technology (Leiden, Netherlands); VIM (ab 16700), prohibitin (PHB; ab55618), CALR (ab2907), heat‐shock cognate 71‐kDa protein (HSP7C; ab19136), PGK1 (ab67335), PRDX4 (ab15574) from Abcam (Cambridge, UK); GRP78 C‐20 (sc‐1051), EZR H‐276 (sc‐20773), β‐actin C4 (sc‐47778), and GAPDH (FL‐335) from Santa Cruz Biotechnology Inc., (Dallas, TX, USA); VINC (10C‐CR1199M1) from Fitzgerald (Acton, MA, USA); ɣH2AX (Ser139) clone JBW301 (#05‐636) from Millipore (Burlington, MA, USA); and heterogeneous nuclear ribonucleoprotein L (HNRPL; A303‐895A) from Bethyl (Montgomery, TX, USA).

Techniques: Inhibition, Knock-Out, Flow Cytometry, Staining, Western Blot, Expressing, Software

Journal: Molecular Oncology

Article Title: HSP90 identified by a proteomic approach as druggable target to reverse platinum resistance in ovarian cancer

doi: 10.1002/1878-0261.12883

Figure Lengend Snippet: Potentiation of CDDP antitumor effect induced by ganetespib in vivo CDDP‐resistant TOV‐112D xenograft model. TOV‐112D Pt‐res pool 2 cells (5 × 10 6 ) were s.c. injected into NSG mice as described in . When tumors were established, mice (five/group) were treated once a week for two weeks, with CDDP (2.5 mg·kg −1 i.p.), ganetespib (GANE; 75 mg·kg −1 i.p.), both drugs in combination, or their respective vehicles (UNT). (A) Relative TV measured at prespecified time points (Means ± SEM). (B) TVs at cutoff when mice were sacrificed. Data are shown as means ± SEM. (C) Mice body weight as surrogate indicator of toxicity for in vivo experiment reported in A. Body weight was measured three times/week. (D) TGD, indicating the mean rate of tumor growth in the treatment groups relative to control untreated mice (see ). Statistically significant results calculated with one‐way ANOVA test are reported (* P < 0.05, ** P < 0.01, and *** P < 0.001). (E) Western blot analysis of γH2AX, cleaved PARP1, cleaved caspase 3, HSP90α, and total HSP90, in lysates from three representative xenograft tumor samples from each treatment group (see ). β‐Actin expression serves as loading control. Western blot quantification was performed by imagej software using the mean value of the three samples for each experimental group.

Article Snippet: Primary antibodies were purchased as follows: HSP90 alpha 2G5.G3 (#SMC‐147) and HSP90 (total) 4F3.E8 (#SMC‐149) from StressMarq Biosciences (Victoria, BC, Canada); GRP75 (#3593), LMN A/C (#2032), ANXA1 (#3299), Bax (#2774), CASPASE 3 (#9662), cleaved PARP1 (Asp214; #5625), PARP1 (#9542), Bcl‐2 (#4223S) from Cell Signaling Technology (Leiden, Netherlands); VIM (ab 16700), prohibitin (PHB; ab55618), CALR (ab2907), heat‐shock cognate 71‐kDa protein (HSP7C; ab19136), PGK1 (ab67335), PRDX4 (ab15574) from Abcam (Cambridge, UK); GRP78 C‐20 (sc‐1051), EZR H‐276 (sc‐20773), β‐actin C4 (sc‐47778), and GAPDH (FL‐335) from Santa Cruz Biotechnology Inc., (Dallas, TX, USA); VINC (10C‐CR1199M1) from Fitzgerald (Acton, MA, USA); ɣH2AX (Ser139) clone JBW301 (#05‐636) from Millipore (Burlington, MA, USA); and heterogeneous nuclear ribonucleoprotein L (HNRPL; A303‐895A) from Bethyl (Montgomery, TX, USA).

Techniques: In Vivo, Injection, Western Blot, Expressing, Software

Journal: PLoS ONE

Article Title: Cardiovascular Protective Effect of Metformin and Telmisartan: Reduction of PARP1 Activity via the AMPK-PARP1 Cascade

doi: 10.1371/journal.pone.0151845

Figure Lengend Snippet: Western blot analysis of AMPK Thr-172 and PARP1 Ser-177 in HUVECs treated with concentrations of H 2 O 2 (A), glucose (B), and Ang II (C) for 4 hr. (D-F) treated with H 2 O 2 (100 μM), glucose (30 mM), or Ang II (100 nM) for the indicated times. Data are mean±SD ratio of phospho-PARP1 Ser-177 to total PARP1 and phospho-AMPK Thr-172 to total AMPK from 3 independent experiments. * p <0.05 compared to controls.

Article Snippet: Rabbit polyclonal anti-phospho-PARP1 Ser-177 antibody was produced with the sequence “ELGFRPEY(pS)ASQ” by AbMax Biotechnology (Beijing, China).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Cardiovascular Protective Effect of Metformin and Telmisartan: Reduction of PARP1 Activity via the AMPK-PARP1 Cascade

doi: 10.1371/journal.pone.0151845

Figure Lengend Snippet: Western blot analysis of AMPK Thr-172 and PARP1 Ser-177 phosphorylation in HUVECs treated with concentrations of metformin (A), glipizide (B), telmisartan (C), and metoprolol (D) for 4 hr or (E-H) metformin (5 mM), glipizide (500 nM), telmisartan (5 μM), and metoprolol (500 μM) for the indicated times. Data are mean±SD ratio of phospho-PARP1 to total PARP1 and phospho-AMPK to total AMPK from 3 independent experiments. * p <0.05 compared to controls.

Article Snippet: Rabbit polyclonal anti-phospho-PARP1 Ser-177 antibody was produced with the sequence “ELGFRPEY(pS)ASQ” by AbMax Biotechnology (Beijing, China).

Techniques: Western Blot, Phospho-proteomics

Journal: PLoS ONE

Article Title: Cardiovascular Protective Effect of Metformin and Telmisartan: Reduction of PARP1 Activity via the AMPK-PARP1 Cascade

doi: 10.1371/journal.pone.0151845

Figure Lengend Snippet: Twelve week-old male C57BL6 mice were administered (A) metformin (200 mg/kg body weight), (B) glipizide (1.3 mg/kg body weight), (C) telmisartan (10 mg/kg body weight), or (D) metoprolol (30 mg/kg body weight) for the indicated times. Control mice received 0.5 ml saline. Western blot analysis of AMPK and PARP1 phosphorylation in aortic extracts from 2 mice pooled. Data are mean ± SD ratio of phospho-PARP1 to total PARP1 and phospho-AMPK to total AMPK (n = 8 mice per group). * p <0.05 compared to controls.

Article Snippet: Rabbit polyclonal anti-phospho-PARP1 Ser-177 antibody was produced with the sequence “ELGFRPEY(pS)ASQ” by AbMax Biotechnology (Beijing, China).

Techniques: Control, Saline, Western Blot, Phospho-proteomics

Journal: PLoS ONE

Article Title: Cardiovascular Protective Effect of Metformin and Telmisartan: Reduction of PARP1 Activity via the AMPK-PARP1 Cascade

doi: 10.1371/journal.pone.0151845

Figure Lengend Snippet: Western blot analysis of protein levels in cell lysates and aortic extracts. (A) HUVECs were infected with Ad-AMPK-CA at 50 or 100 multiplicities of infection (MOI) or Ad-null virus at 50 MOI for 24 hr. (B,C) HUVECs were pre-treated with or without AICAR (1 mM) for 30 min before the addition of glucose (30 mM) or Ang II (100 nM) at the indicated concentrations for 4 hr. (D) HUVECs were pre-treated with or without Compound C (15 μM) for 30 min before metformin (5 mM) for 4 hr. (E) AMPKα2 +/+ and AMPKα2 -/- mice were orally administered with or without metformin (200 mg/kg body weight) and aortas were collected after 12 hr. Data are mean±SD ratio of phospho-PARP1 to total PARP1 and phospho-AMPK to total AMPK from at least 3 experiments in A-D and n = 8 animals in E. * p <0.05 compared with controls.

Article Snippet: Rabbit polyclonal anti-phospho-PARP1 Ser-177 antibody was produced with the sequence “ELGFRPEY(pS)ASQ” by AbMax Biotechnology (Beijing, China).

Techniques: Western Blot, Infection, Virus

Journal: PLoS ONE

Article Title: Cardiovascular Protective Effect of Metformin and Telmisartan: Reduction of PARP1 Activity via the AMPK-PARP1 Cascade

doi: 10.1371/journal.pone.0151845

Figure Lengend Snippet: (A-F) Western blot analysis of protein PAR in EC lysates. (A, B) HUVECs were pre-treated with or without AICAR for 4 hr before the addition of glucose (30 mM) (A) or Ang II (100 nM) (B) and incubated for another 24 hr. (C) HUVECs were treated with or without metformin or glipizide for 6 hr, then incubated with or without 30 mM glucose for 24 hr. (D) HUVECs were treated with or without telmisartan or metoprolol for 6 hr, then incubated with or without 100 nM Ang II for 24 hr. (E, F) BAECs were transfected with flag-tagged wild-type (WT), S177A, or S177D PARP1 plasmids for 24 hr, then incubated with 30 mM glucose (E) or 100 nM Ang II (F) for 6 hr. (G) BAECs were transfected with S177A or S177D PARP1 plasmid. RT-PCR analysis of mRNA level of eNOS, SIRT1, KLF4, MCP-1, and VCAM-1. (H, I) BAECs were transfected with S177A or S177D PARP1 or WT PARP1 treated with or without PJ-34 (3 μM for 6 hr). (H) Western blot analysis and quantification of protein levels. (I) Measurement of PARP1 activity in nuclear extracts of BAECs and NO level in the cultured medium. Data are mean±SD from at least 3 experiments. * p <0.05 compared with controls.

Article Snippet: Rabbit polyclonal anti-phospho-PARP1 Ser-177 antibody was produced with the sequence “ELGFRPEY(pS)ASQ” by AbMax Biotechnology (Beijing, China).

Techniques: Western Blot, Incubation, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Activity Assay, Cell Culture

Journal: PLoS ONE

Article Title: Cardiovascular Protective Effect of Metformin and Telmisartan: Reduction of PARP1 Activity via the AMPK-PARP1 Cascade

doi: 10.1371/journal.pone.0151845

Figure Lengend Snippet: db/m and db/db mice were treated with metformin (200 mg/kg/day) or glipizide (1.3 mg/kg/day) for 2 weeks. SHR and WKY rats were treated with telmisartan (10 mg/kg/day) or metoprolol (30 mg/kg/day) for 8 weeks. (A, D) Western blot analysis of protein levels in aortic extracts from various animal groups were analyzed by western blotting with various antibodies as indicated. (B, E) Scatter plots of ratio of phospho-PARP1 to total PARP1, phospho-AMPKα to total AMPKα, and PAR to β-actin for each aortic specimen. (C, F) RT-PCR analysis of mRNA level of eNOS, SIRT1, KLF4, ICAM-1, and VCAM-1 in rodent aortas. * p <0.05 compared with controls.

Article Snippet: Rabbit polyclonal anti-phospho-PARP1 Ser-177 antibody was produced with the sequence “ELGFRPEY(pS)ASQ” by AbMax Biotechnology (Beijing, China).

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal:

Article Title: Poly(ADP-Ribose) Polymerase 1 Binds to Kaposi's Sarcoma-Associated Herpesvirus (KSHV) Terminal Repeat Sequence and Modulates KSHV Replication in Latency

doi: 10.1128/JVI.78.18.9936-9946.2004

Figure Lengend Snippet: Western blot analysis of the TR(+)Sepharose bound and unbound fractions of BC3 and BJAB cell extracts. (A) Comparison of factors bound to the TR(+)Sepharose column. An aliquot of cell extract (CE; 0.1%), pass-through (Th; 0.01%), and KCl-eluted fractions of BC3 and BJAB cells were separated by SDS-4 to 20% PAGE, blotted on a polyvinylidene difluoride membrane, and analyzed with the commercially available antibodies shown to the left of the panel (α, anti). Typically detected bands are indicated by a bracket for LANA and arrows for ORC2, CDC6, Mcm7, and MSH6. (B) Comparison of bound and unbound fractions to TR(+)Sepharose and K-oriLyt CS Sepharose. K-oriLyt CS was also tested for TPA-uninduced or -induced materials. Binding of PARP1 and MSH6 to TR and K-oriLyt CS was examined. +, present; −, absent.

Article Snippet: The anti-PARP1 antibody was a mouse monoclonal antibody (MBL), and the anti-LANA antibody was a rabbit polyclonal antibody (a gift from T. Sata) ( 31 ).

Techniques: Western Blot, Comparison, Membrane, Binding Assay

Journal:

Article Title: Poly(ADP-Ribose) Polymerase 1 Binds to Kaposi's Sarcoma-Associated Herpesvirus (KSHV) Terminal Repeat Sequence and Modulates KSHV Replication in Latency

doi: 10.1128/JVI.78.18.9936-9946.2004

Figure Lengend Snippet: PARP1 is colocalized with LANA. Anti-PARP1 (Alexa 546, red), anti-LANA (Alexa 488, green), and DAPI (blue) were used to stain nuclei, and merged images are also shown for each case. All of the data were obtained with a Zeiss 510 laser confocal fluorescent microscope. BC3 cells (A), BJAB cells (B), and 293 cells (C) expressing LANA and containing multiple copies of BAC TR-2, a TR-containing bacterial artificial chromosome construct.

Article Snippet: The anti-PARP1 antibody was a mouse monoclonal antibody (MBL), and the anti-LANA antibody was a rabbit polyclonal antibody (a gift from T. Sata) ( 31 ).

Techniques: Staining, Microscopy, Expressing, Construct

Journal:

Article Title: Poly(ADP-Ribose) Polymerase 1 Binds to Kaposi's Sarcoma-Associated Herpesvirus (KSHV) Terminal Repeat Sequence and Modulates KSHV Replication in Latency

doi: 10.1128/JVI.78.18.9936-9946.2004

Figure Lengend Snippet: PARP1-binding assay. (A) Schematic representation of the TR fragments used for PARP1-binding experiments with ELISA and EMSA. Constructs 1, 2, 2-1, 2-1-1, and 2-1-2 were used in ELISAs, and constructs 2-2 to 2-5 were used in EMSAs. The position of the LANA-binding sequence (LBS) and the suspected PARP1-binding sequences (5′-TCGNT-3′) reported by Nirodi et al. (41) are also shown. (B) PARP1 binding by ELISA. The bound biotinylated fragment was detected colorimetrically by using a streptavidin-biotin-AP complex and pNpp as a substrate. The optical density at 405 nm was measured, and the arbitrary scale of binding intensity for each fragment is shown, where the intensity for the full-length TR was set at 1. (C) EMSA. Each fragment was fill-in labeled with Klenow fragment and [α-32P]dCTP. Lanes 1, 4, 7, and 10, probe only; lanes 2, 5, 8, and 11, probes were incubated with 100 ng of human PARP1 (Trevigen); lanes 3, 6, 9, and 12, each probe was incubated with 100 ng of human PARP1 and a 100-fold excess of each nonlabeled probe. Specifically shifted bands are indicated by arrowheads. (D) EMSA with specific antibodies against PARP1. In this analysis, constructs 2-2 and 2-3 were analyzed. Lanes 1 and 6, probes only; lanes 2 and 7, probes were incubated with 100 ng of human PARP1; lanes 3 and 8, probes were incubated with rabbit polyclonal anti-PARP1 (αPARP1) antibodies (2 μg) and 100 ng of human PARP1; lanes 4 and 9, probes were incubated with normal rabbit IgG (2 μg) and 100 ng of human PARP1; lanes 5 and 10, probes were incubated with 100 ng of human PARP1 and a 100-fold excess of nonlabeled probes. The band that specifically shifted and disappeared with the addition of antibody is indicated by an arrowhead. Asterisks denote nonspecific binding. +, present; −, absent.

Article Snippet: The anti-PARP1 antibody was a mouse monoclonal antibody (MBL), and the anti-LANA antibody was a rabbit polyclonal antibody (a gift from T. Sata) ( 31 ).

Techniques: Binding Assay, Enzyme-linked Immunosorbent Assay, Construct, Sequencing, Labeling, Incubation

Journal:

Article Title: Poly(ADP-Ribose) Polymerase 1 Binds to Kaposi's Sarcoma-Associated Herpesvirus (KSHV) Terminal Repeat Sequence and Modulates KSHV Replication in Latency

doi: 10.1128/JVI.78.18.9936-9946.2004

Figure Lengend Snippet: Poly(ADP-ribosyl)ation assay of LANA. (A) Immunoprecipitation followed by Western blot analysis. Whole-cell extracts (WCE) from BC3 cells were immunoprecipitated (IP) with the designated antibodies (2 μg of each) and immunoblotted (IB) with a rat monoclonal anti-LANA antibody (left) or rabbit polyclonal anti-PAR antibodies (right). Multiple forms of LANA are indicated. The arrowhead shows poly(ADP-ribosyl)ated LANA detected by the PAR antibodies. (B) Biotinylated NAD incorporation assay. LANA was immunoprecipitated with either a rat monoclonal anti-LANA (αLANA) antibody (2 μg) or normal rat IgG (2 μg). The immunoprecipitates were incubated with biotinylated NAD and 100 ng of purified human PARP1 (Trevigen) as described in Materials and Methods. The samples were then fractionated by SDS-PAGE and blotted with either a rat monoclonal anti-LANA antibody (2 μg) (left) or streptavidin-HRP (right). The immunoprecipitated LANA (left) and NAD-incorporated LANA and PARP1 are indicated by arrowheads. +, present; −, absent.

Article Snippet: The anti-PARP1 antibody was a mouse monoclonal antibody (MBL), and the anti-LANA antibody was a rabbit polyclonal antibody (a gift from T. Sata) ( 31 ).

Techniques: Immunoprecipitation, Western Blot, Incubation, Purification, SDS Page

Journal:

Article Title: Poly(ADP-Ribose) Polymerase 1 Binds to Kaposi's Sarcoma-Associated Herpesvirus (KSHV) Terminal Repeat Sequence and Modulates KSHV Replication in Latency

doi: 10.1128/JVI.78.18.9936-9946.2004

Figure Lengend Snippet: Viral genome copy number and cell viability in BC3 cells treated with drugs affecting PARP activity. BC3 cells were untreated (NT) or treated with NA or HU as described in Materials and Methods and tested for viral genome copy number by real-time PCR (A) and for cell viability by the trypan blue method (B). (A) Viral genome copy number in drug-treated cells. The viral genome copy number was estimated by amplifying the K-oriLyt CS region and normalized to the β-globin gene content by real-time PCR on the designated day. The number of copies is shown on a logarithmic scale as viral copy number per picogram of DNA. The value above the bar shows a mean value for each case. (B) Cell viability of dru-treated cells. The viable cells were counted on the designated day as described. (C) HU treatment leads to the increase in poly(ADP-ribosyl)ation of LANA. BC3 cells were treated with NT (nontreatment), TPA (25 ng/ml; Sigma), HU (50 μM), or NA (10 mM), and 3 days after treatment, cells were harvested and solubilized with RIPA buffer and the soluble fraction was immunoprecipitated (IP) with either a preimmune rat IgG or a rat monoclonal anti-LANA antibody. The immunoprecipitated fraction was fractionated by SDS-PAGE and immunoblotted (IB) with either rabbit polyclonal anti-PAR (αPAR) antibodies or a rat monoclonal anti-LANA (αLANA) antibody. (D) No lytic gene is expressed by drug treatment. BC3 cells were fixed with 4% paraformaldehyde-PBS 1 day after treatment with NT (nontreatment), TPA (25 ng/ml; Sigma), HU (50 μM), or NA (10 mM). Then the cells were incubated with a mouse monoclonal anti-RTA antibody, followed by goat anti-mouse IgG conjugated with Alexa 488 (Molecular Probes-Invitrogen) (green signal). The signal was detected with a confocal fluorescence microscope (Zeiss 510) (original magnification, ×600). The nucleus was counterstained with DAPI.

Article Snippet: The anti-PARP1 antibody was a mouse monoclonal antibody (MBL), and the anti-LANA antibody was a rabbit polyclonal antibody (a gift from T. Sata) ( 31 ).

Techniques: Activity Assay, Real-time Polymerase Chain Reaction, Immunoprecipitation, SDS Page, Incubation, Fluorescence, Microscopy