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: Journal of the American Society of Nephrology

Article Title: Negative Modulation of B Cell Activation by Melanocortin 1 Receptor Signaling Protects against Membranous Nephropathy

doi: 10.1681/asn.2022050605

Figure Lengend Snippet: Figure 8. Loss of MC1R potentiates B-cell activation and en- hances autologous humoral immune responses in rats with PHN. (A) Animals were treated as elaborated in Figure 1F. Splenocytes derived from WT or KO rats with PHN were pro- cessed for flow cytometry analysis of CD1381 cells. (B) Arbitrary levels of sheep IgG-specific rat IgG in sera collected from WT or KO rats with PHN, as estimated by ELISA of diluted sera; aP,0.05 versus WT rats with PHN (n56). (C) Schematic diagram depicting the working model for negative regulation of B-cell activation by MC1R-mediated melanocortinergic signaling. As a typical G-protein coupled receptor, MC1R is expressed by B lympho- cytes and is able to trigger the cAMP signaling pathway on ac- tivation by diverse melanocortins, such as pan-MCR agonists like ACTH and NDP-MSH, or the selective MC1R agonist MS05. Subsequently, cAMP signaling downstream of MC1R activates MITF, one of the major signaling transducers of MC1R pathway and a critical regulator of a plethora of key cellular processes, including melanogenesis in melanocytes and cellular differenti- ation in nonmelanocytic cells. In B lymphocytes, MITF acts as a repressor of IRF4, a characteristic lymphoid transcription factor essential for B-cell development and maturation. MC1R-medi- ated melanocortinergic signaling suppresses B-cell activation and plasmacytic differentiation and reduces IgG production via the cAMP/MITF/IRF4 pathway, which could be intercepted by SQ22536, an adenylyl cyclase inhibitor. This MC1R-mediated B- cell regulatory signaling is defective in B cells with MC1R KO and could be partially restored by the cAMP mimetic dBcAMP.

Article Snippet: IgG levels in cell culture supernatants were detected by using a rat IgG ELISA kit (Aviva Systems Biology, San Diego, CA) according to the manufacturer’s instruction.

Techniques: Activation Assay, Derivative Assay, Cytometry, Enzyme-linked Immunosorbent Assay

Journal: PLoS Pathogens

Article Title: Plasminogen Controls Inflammation and Pathogenesis of Influenza Virus Infections via Fibrinolysis

doi: 10.1371/journal.ppat.1003229

Figure Lengend Snippet: (A) Histopathological analysis of lungs from infected WT and PLG-KO mice inoculated with A/PR/8/34 virus (day 3 post-infection) or A/Netherlands/602/09 virus (day 5 post-infection). Thin sections of lungs obtained from infected and uninfected WT and PLG-KO mice (as indicated) were stained with hematoxilin end eosin (HE) to evaluate histopathological changes. Note the marked infiltration of inflammatory cells in the lungs of infected WT mice, which was largely absent in the lungs of PLG-KO mice. The results shown are representative for two-three mice for both groups. Immunohistochemistry (IHC) using a monoclonal antibody for the influenza A virus nucleoprotein was used to detect virus-infected cells. Cells positive for the presence of viral antigen stained red. (B) Cytokine levels in BAL were assessed by ELISA on the indicated days post inoculation of WT (black bars) and PLG-KO mice (white bars) with IAV A/PR/8/34 or A/Netherlands/602/09. Data represent mean ± s.e.m. of 3–6 mice per group.

Article Snippet: Viruses, cells, and reagents used, were: IAV A/Netherlands/602/09 , A/chicken/Ivory-Coast/1787/2006 , A/PR/8/34 (American Type Culture Collection, ATCC), A549 cells (ATCC), Madin-Darby Canine Kidney cells (MDCK, ATCC), trypsin (Becton Dickinson), plasminogen and 6-AHA (Sigma), Ancrod (NIBSC), 23-Plex Mouse Cytokine Assay (Bio-Rad), ELISA kits for mouse -IL-6, -KC, -–RANTES, -IFN-α -IFN-γ(R&D Systems), -plasminogen (Mybiosource), -active plasmin (Kordia), -D-dimer, -fibrinogen and -FDP (Genway), antibodies anti-HA (Santa Cruz), anti-tubulin (Sigma), anti-NP (ATCC), anti-fibrinogen (Genway).

Techniques: Infection, Virus, Staining, Immunohistochemistry, Enzyme-linked Immunosorbent Assay

Journal: PLoS Pathogens

Article Title: Plasminogen Controls Inflammation and Pathogenesis of Influenza Virus Infections via Fibrinolysis

doi: 10.1371/journal.ppat.1003229

Figure Lengend Snippet: (A) Levels of Plasminogen, Active Plasmin, FDP, D-dimer and Fibrinogen, were determined by ELISA in the BAL of A/PR/8/34 infected or uninfected (−) C57BL/6 mice after the indicated days post-inoculation. Markers were also evaluated in the BAL of WT or PLG-KO mice infected with A/Netherlands/602/09 virus. Data represent mean ± s.e.m of n = 3–6 mice per group. (B) Western blot analysis for the detection of fibrinogen and FDP in the lungs of IAV-infected mice on the indicated days post inoculation (representative of n = 3). kDa: apparent molecular weight. n = mice per group. (C) Presence of fibrinogen was assessed in the blood of mice treated or not with Ancrod by ELISA (left panel) or Western blot analysis (right panel). The results represent the mean values ± s.e.m from 3 individual animals per group for the ELISA. The western blot analysis is representative for results of 3 mice per group.

Article Snippet: Viruses, cells, and reagents used, were: IAV A/Netherlands/602/09 , A/chicken/Ivory-Coast/1787/2006 , A/PR/8/34 (American Type Culture Collection, ATCC), A549 cells (ATCC), Madin-Darby Canine Kidney cells (MDCK, ATCC), trypsin (Becton Dickinson), plasminogen and 6-AHA (Sigma), Ancrod (NIBSC), 23-Plex Mouse Cytokine Assay (Bio-Rad), ELISA kits for mouse -IL-6, -KC, -–RANTES, -IFN-α -IFN-γ(R&D Systems), -plasminogen (Mybiosource), -active plasmin (Kordia), -D-dimer, -fibrinogen and -FDP (Genway), antibodies anti-HA (Santa Cruz), anti-tubulin (Sigma), anti-NP (ATCC), anti-fibrinogen (Genway).

Techniques: Enzyme-linked Immunosorbent Assay, Infection, Virus, Western Blot, Molecular Weight

Journal: PLoS Pathogens

Article Title: Plasminogen Controls Inflammation and Pathogenesis of Influenza Virus Infections via Fibrinolysis

doi: 10.1371/journal.ppat.1003229

Figure Lengend Snippet: (A) Survival and weight loss of mice treated with Ancrod (open symbols, n = 11) or not (closed symbols, n = 11) after infection with IAV A/PR/8/34 (squares) or uninfected mice (diamonds, n = 5). Weight loss data represent weight average ± s.e.m of the above indicated number of mice. (B) Cytokines levels in the BAL were measured by ELISA after A/PR/8/34 infection of WT and PLG-KO (KO) mice treated with Ancrod (white bars) or not (black bars). Data represent mean ± s.e.m. of n = 4 mice per group. (C) Survival rate (left panels) and weight loss (right panels) of WT (squares) and PLG-KO (triangles) mice treated with Ancrod (open symbols) or not (closed symbols) after intranasal inoculation with IAV A/PR/8/34 (n = 8–10 mice per group). Weight loss data represent weight average ± s.e.m of the above indicated number of mice.

Article Snippet: Viruses, cells, and reagents used, were: IAV A/Netherlands/602/09 , A/chicken/Ivory-Coast/1787/2006 , A/PR/8/34 (American Type Culture Collection, ATCC), A549 cells (ATCC), Madin-Darby Canine Kidney cells (MDCK, ATCC), trypsin (Becton Dickinson), plasminogen and 6-AHA (Sigma), Ancrod (NIBSC), 23-Plex Mouse Cytokine Assay (Bio-Rad), ELISA kits for mouse -IL-6, -KC, -–RANTES, -IFN-α -IFN-γ(R&D Systems), -plasminogen (Mybiosource), -active plasmin (Kordia), -D-dimer, -fibrinogen and -FDP (Genway), antibodies anti-HA (Santa Cruz), anti-tubulin (Sigma), anti-NP (ATCC), anti-fibrinogen (Genway).

Techniques: Infection, Enzyme-linked Immunosorbent Assay

Journal: STAR Protocols

Article Title: Protocol to study sufficiency of cis -regulatory elements in mouse embryonic stem cells using a CRISPR-mediated knockin approach

doi: 10.1016/j.xpro.2022.101492

Figure Lengend Snippet:

Article Snippet: Wide Mini-Sub Cell GT Horizontal Electrophoresis System , Bio-Rad , Cat: 1704468.

Techniques: Virus, Recombinant, Knock-Out, Polymerase Chain Reaction, Transfection, Plasmid Preparation, Sequencing, Cloning, Software, CRISPR, Electrophoresis