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 , The phosphorylation of ATM(Ser 1987), ATM whole protein in HEI-OC1 cells treated with MA-5 S- enantiomer at 0, 3,10, 30 μM for 24hr. b , The SIRT1 and SRIT3 protein expression in HEI-OC1 cells treated with S-enantiomer at 0 and 30 μM with or without ATM inhibitor KU-60019 at 10μM for 24hr. c , Phosphorylation of TRIM28(Ser 824), Phosphorylation of TRIM28(Ser 473) TRIM28 hole protein and ATM protein expression in in HEI-OC1 cells after ATM siRNA knock down. Right panel, the quantitative analysis of TRIM28(Ser 824) phosphorylation. * p <0.05, ** p <0.01 vs. control; statistics were calculated byOne way anova Turkey’s multiple comparison test. d , Phosphorylation of p53(Ser 15) expression and p53 whole protein in HEI-OC1 cells treated with MA-5 racemate at 0, 1, 3,10, 30 μM for 24hr. e , The phosphorylation of DNA-PKcs(Ser 2056) and DNA-PKcs whole in HEI-OC1 cells treated with S-enantiomer with or without DNA-PK inhibitor, M3814 (10nM). f , The phosphorylation of TRIM28(Ser 824) and TRIM28 hole protein and DNA-PKcs hole protein expression in in HEI-OC1 cells after DNA-PKcs siRNA knock down procedure. Control (Lipofectamine-RNAiMAX regent alone without siRNA), Negative-Control (NC) siRNA transfected HEI-OC1cells were incubated for 48hr and treated with MA-5 S enantiomer at 30 μM for another 24hr. Data were mean ± SE. * p <0.05, ** p <0.01 vs. DMSO control; statistics were calculated by One way anova Turkey’s multiple comparison. g , DNA-PK inhibitor reduced the S-enantiomer-induced SIRT1 expression. The protein expression level of all SIRT1-SIRT7 in HEI-OC1 cells treated with 0.1 % DMSO or DNA-PK inhibitor M3814 at 10nM for 24hr. Data were mean ± SE. * p <0.05, ** p <0.01 vs. DMSO control; statistics were calculated by unpaired two-side t-test. h , The he protein expression of SIRT1-7 when SIR2, SIRT5, and Sirt6 were knocked down. Data were mean ± SE. * p <0.05, ** p <0.01 vs. DMSO control; statistics were calculated by one way anova Turkey’s multiple comparison.

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: Phospho-proteomics, Expressing, Knockdown, Control, Comparison, Negative Control, Transfection, Incubation