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

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: Cellular microbiology

Article Title: Porphyromonas gingivalis influences actin degradation within epithelial cells during invasion and apoptosis.

doi: 10.1111/j.1462-5822.2012.01780.x

Figure Lengend Snippet: Fig. 3. P. gingivalis hydrolyse b-actin. HGECs were challenged with P. gingivalis 33277 and W50 at moi:100 with or without human serum up to 48 h and total proteins were collected at indicated time points. Twenty micrograms each of total protein was immunoblotted against anti-human b-actin. P. gingivalis 33277 (A) and W50 (C) induced b-actin hydrolysis as early as 1 h whereas HGECs treated with human serum protected b-actin degradation (B and D). HGECs were challenged with P. gingivalis 33277 for indicated time points and immunoblotted against human b-actin antibody and time-dependent actin hydrolysis by P. gingivalis was noted (E). Serum alone did not have any effect on b-actin (F). In a different experiment, HGECs were treated with P. gingivalis 33277 for 24 h and immunoblotted against PARP antibody. The immunoblot shows PARP cleavage product of 23 kDa which is characteristic of apoptosis (G). The time-course experiment was performed to determine the extent of apoptosis by P. gingivalis using cell death detection ELISA. HGECs were pre-treated either with irreversible pan-caspase inhibitor (Z-VAD-FMK) or jasplakinolide or with vehicle alone for 2 h and then challenged with P. gingivalis for indicated time point. Total proteins collected at each time point was subjected cell death detection ELISA. The time-dependent induction of apoptosis in HGECs by P. gingivalis was noted as increased in optical density (H). GAPDH was used as loading control in all the blots. All the experiments were repeated at least three times and representative blots are shown.

Article Snippet: PARP antibody (AF-600-NA) was procured from R&D Systems.

Techniques: Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: Science Advances

Article Title: TAp63 determines the fate of oocytes against DNA damage

doi: 10.1126/sciadv.ade1846

Figure Lengend Snippet: ( A to C ) IF assays of γH2AX (A), BAX (B), and cPARP (C) expression in the ovaries from wild-type and Trp63 cKO female mice at 6, 13, 18, and 24 hours after CPA injection. Scale bars, 50 μm. ( D ) Percentage of primordial oocytes with BAX (top) and cPARP (bottom) expression. ( E ) IF assays of BAX (top) and cPARP (bottom) expression in the ovaries from wild-type and Trp63 cKO mice treated with solvent or DOXO for 24 hours. Scale bars, 50 μm. ( F ) Transmission electron microscopy (TEM) images of gross primordial follicles, nucleus, and mitochondria from CD-1 mice at 18 hours after phosphate-buffered saline (PBS), DOXO, and CPA injection. NM, nucleus membrane; Pre-G, pregranulosa cell; N, nucleus; Cyto, cytoplasm; M, mitochondria; C, cristae of mitochondria. ( G ) IF assays of OPA1 expression in the ovaries from wild-type and Trp63 cKO female mice at 6, 13, 18, and 24 hours after CPA injection. Insets show high magnification of primordial follicles with the expression of OPA1. Scale bars, 50 μm. ( H ) Relative intensity of OPA1 in primordial follicles of the ovaries from wild-type and Trp63 cKO female mice at 6, 13, 18, and 24 hours after CPA injection. The numbers of oocytes measured for the intensity of OPA1 were counted. ( I ) Schematic of TAp63-regulated apoptosis in oocytes of primordial follicles.

Article Snippet: The catalog numbers and dilution of primary antibodies were as follows: p63 (D9L7L) (39692s; 1:100), γH2AX (9718S; 1:100), cPARP (9548s; 1:50), p-CHK2 (2197s; 1:50), and OPA1 (D7C1A) (67589s; 1:50) from Cell Signaling Technology; BAX (p-19) (sc-526; 1:50) from Santa Cruz Biotechnology Inc.; and DDX4 (ab270534; 1:100) and FOXL2 (ab246511; 1:50) from Abcam.

Techniques: Expressing, Injection, Solvent, Transmission Assay, Electron Microscopy, Saline, Membrane