Fig. 4: Disruption of DNA damage repair activity by cyclic peptides.

a The chemical composition of cyclic peptide 2 and its TAMRA labeled 26. b Representative confocal images of 26 in live U2OS cells. Images representative of n = 2 biologically independent experiments. Scale bars 20 μm. c Western blot analysis of lysates from U2OS cells treated with 33 (a scrambled sequence of cyclic peptide 2) and 2 (upper panels). H2AX was used as a loading control (lower panels) which was performed in a different blot. Representative of n = 3 independent experiments. d Quantified relative γ-H2AX signals (c) for n = 3 independent experiments. Data are presented as mean values ± SD. (Two-tailed, unpaired t-test *p = 0.0112 and ns is non-significant). (DMSO: gray, 33: yellowish-green, and 2: skyblue). e Western blot analysis of lysates from U2OS cells treated with 2 and 20 (upper panels). H2AX was used as a loading control (lower panels) which was performed in a different blot. Representative image of n = 3 independent experiments. f Quantified relative γ-H2AX signal from (e) for n = 3 independent experiments. Data are presented as mean values ± SD. (Two-tailed, unpaired t-test ***p = 0.0003 and **p = 0.003). (DMSO: gray, 2: skyblue, and 20: salmon). g DNA damage was visualized by a “comet-like” vista green signal from the DNA of individual cells, analyzed over 100 cells in n = 2 independent experiments. Scale bars 10 μm. h Quantified relative tail moment of images (g) for n = 2 independent experiments, using “OpenComet” plugged-in to FiJi (ImageJ, open version). Data are presented as mean values (DMSO: gray, 2: skyblue, and 20: salmon). i Western blot using anti-flag antibody (upper panels) and anti-ubiquitin(Lys63-specific) (lower panels) from anti-flag immunoprecipitated 293T cell lysates treated with and without 2 and transfected with RFN168 wt or mutant. Representative of n = 2 independent experiments. j The cell cycle distribution of HeLa cells treated with and without 2 after 72 and 96 h. From n = 2 independent flow cytometry experiments (>15,000 cells each condition). Data are presented as mean values (DMSO: silver, 2 (72 h): bluish green, and 2 (96 h): orange). k The relative population of annexin V-FITC (apoptotic) cells after 96 h. From n = 2 independent flow cytometry experiments (>20,000 cells for each condition). Data are presented as mean values (DMSO: gray and 2: skyblue). All source data are provided as a Source data file.