Journal: NPJ Aging

Article Title: Human fibroblasts from aged individuals exhibit chromosomal instability through replication stress caused by oxidative stress

doi: 10.1038/s41514-025-00299-w

Figure Lengend Snippet: A Number of γ-H2AX foci. Cells were fixed and stained with an antibody against γ-H2AX. Representative images are shown. 15–24 cells for each cell were analyzed and displayed as box and dot plots in the right graph. Data from different cells are shown in different colors. P -value was obtained using the Mann-Whitney U -test. B Rates of cells with 53BP1 nuclear bodies. Cells were fixed and stained with an antibody against 53BP1. Representative images were shown (arrowheads). 95–138 cells for each cell were analyzed. P -value was obtained using the Welch’s t -test. C Rates of cells with ultrafine bridges. Cells were treated with RO-3306 for 12 h, released and fixed after 1 h, and stained with an antibody against PICH (ERCC6L). A representative image is shown (arrowheads). 46–66 cells were counted for each cell. P -value was obtained using the Student’s t -test. D Chromosome breaks were observed in 50–105 metaphase chromosome spreads for each cell. A representative image is shown (arrowhead). Percentages of cells with chromosome breaks were shown in the right graph. P -value was obtained using the Student’s t -test. E Replication fork speed. Cells were sequentially pulse-labeled with digoxigenin-conjugated deoxyuridine and biotin-conjugated deoxyuridine. Representative fibers are shown. Fork speeds were determined through the length of both labels (digoxigenin-dUTPs + biotin-dUTPs), according to the previous report that 1 μm is approximately equal to 3.5 kb of DNA . 101–138 fibers per cell were quantified and shown as box and dot blots. P -value was obtained using the Mann–Whitney U test. F Replication fork asymmetry. Cells were treated as in (E). Representative fibers are shown. Fork asymmetry was determined by dividing longer biotin-dUTPs label on a bi-directional replication fork with shorter biotin-dUTPs label for 49-79 fibers per cell and shown as box and dot blots. P -value was obtained using the Mann–Whitney U test. In all the graphs, red, blue, or black-filled circles represent skin fibroblasts, while yellow-filled circles represent lung fibroblasts. Error bars represent S.D. DNA was stained with DAPI. Scale bars: 5 μm.

Article Snippet: For nucleoside supplementation, a mix of deoxycytidine (Tokyo Chemical Industry, D3583), deoxyadenosine (Tokyo Chemical Industry, D0046), thymidine (Tokyo Chemical Industry, T0233) and deoxyguanosine (Tokyo Chemical Industry, D0052) was applied at 20 μM for 48 h. Rabbit polyclonal antibodies for 53BP1 (NOVUS Biologicals, NB100-304) and PICH (ERCC6L; Proteintech, 15688-1-AP) were used for immunofluorescence at 1:1,000.

Techniques: Staining, MANN-WHITNEY, Labeling

Journal: NPJ Aging

Article Title: Human fibroblasts from aged individuals exhibit chromosomal instability through replication stress caused by oxidative stress

doi: 10.1038/s41514-025-00299-w

Figure Lengend Snippet: A Number of γ-H2AX foci in the presence or absence of NAC. Cells were treated with NAC for 48 h, fixed, and stained with an antibody against γ-H2AX. Representative images are shown. 33-49 cells were analyzed for each cell and displayed as box and dot plots in the right graph. P -values were obtained using the Steel-Dwass multiple comparison test. B Rates of cells with 53BP1 nuclear bodies in the presence or absence of NAC. Cells were treated with NAC for 48 h, fixed, and stained with an antibody against 53BP1. 526-770 cells were analyzed for each cell. P -values were obtained using the Tukey–Kramer multiple comparison test. C Rates of cells with ultrafine bridges in the presence or absence of NAC. Cells were treated with NAC for 48 h and stained with an antibody against PICH (ERCC6L). DNA was stained with DAPI. 72-103 cells were counted for each cell. P -values were obtained using the Tukey–Kramer multiple comparison test. D Rates of cells with 53BP1 nuclear bodies in cells supplemented with or without nucleosides (NUC). Cells were supplemented with nucleosides for 48 h, fixed, and stained with an antibody against 53BP1. 401-890 cells were counted for each cell. P -values were obtained using the Tukey–Kramer multiple comparison test. E Rates of cells with ultrafine bridges in cells supplemented with or without nucleosides. Cells were treated as in ( D ). Then cells were fixed and stained with an antibody against PICH (ERCC6L). DNA was stained with DAPI. 39-70 cells were counted for each cell. P -values were obtained using the Tukey–Kramer multiple comparison test. F Micronucleation rates of cells supplemented with or without nucleosides. Cells were treated as in ( D ). Then cells were fixed and stained with DAPI. 521-741 cells were counted for each cell. P -values were obtained using the Tukey–Kramer multiple comparison test. In all the graphs, red, blue, or black-filled circles represent skin fibroblasts, while yellow-filled circles represent lung fibroblasts. Error bars represent S.D. n.s., not statistically significant. Scale bars: 5 μm.

Article Snippet: For nucleoside supplementation, a mix of deoxycytidine (Tokyo Chemical Industry, D3583), deoxyadenosine (Tokyo Chemical Industry, D0046), thymidine (Tokyo Chemical Industry, T0233) and deoxyguanosine (Tokyo Chemical Industry, D0052) was applied at 20 μM for 48 h. Rabbit polyclonal antibodies for 53BP1 (NOVUS Biologicals, NB100-304) and PICH (ERCC6L; Proteintech, 15688-1-AP) were used for immunofluorescence at 1:1,000.

Techniques: Staining, Comparison

Journal: NPJ Aging

Article Title: Human fibroblasts from aged individuals exhibit chromosomal instability through replication stress caused by oxidative stress

doi: 10.1038/s41514-025-00299-w

Figure Lengend Snippet: A Cells were synchronized by RO-3306 for 12 h, released and treated with MG132 for 3 h, and either fixed directly or after cold treatment for 10 min. Cells were stained with an antibody against α-tubulin, and relative fluorescence intensity on the spindle at 10 min compared with that at 0 min was quantified for 5–7 cells for each cell. The average value of four young or old individuals at the time 0 is set to 1. P -value was obtained using the Student’s t -test. B Cells were treated with NAC for 48 h. Relative fluorescence intensity was quantified as in ( A ) for 10-24 cells for each cell. C Cells were supplemented with nucleosides for 48 h. Relative fluorescence intensity was quantified as in ( A ) for 10-27 cells for each cell. D Cells were treated with UMK57 for 24 h. Relative fluorescence intensity was quantified as in (A) for 10-28 cells for each cell. E Cells were treated with UMK57 for 24 h, fixed, and stained with DAPI. 251-534 cells were counted for each cell. The average value of four young or old individuals at the time 0 is set to 1. F Cells were treated as in ( D ), fixed, and stained with an antibody against γ-H2AX. 49-70 cells were analyzed for each cell and displayed as box and dot plots. P -values were obtained using the Steel-Dwass multiple comparison test. G Cells were treated as in ( D ), and fixed and stained with an antibody against 53BP1. 261-416 cells were analyzed for each cell. H Schematic of the mechanism of CIN in fibroblasts from aged individuals suggested in this study. See text for details. In all the graphs, red, blue, or black-filled circles represent skin fibroblasts, while yellow-filled circles represent lung fibroblasts. In ( B ), ( C ), ( D ), ( E ), and ( G ), P -values were obtained using the Tukey–Kramer multiple comparison test. Error bars represent S.D. n.s., not statistically significant. Scale bars: 5 μm.

Article Snippet: For nucleoside supplementation, a mix of deoxycytidine (Tokyo Chemical Industry, D3583), deoxyadenosine (Tokyo Chemical Industry, D0046), thymidine (Tokyo Chemical Industry, T0233) and deoxyguanosine (Tokyo Chemical Industry, D0052) was applied at 20 μM for 48 h. Rabbit polyclonal antibodies for 53BP1 (NOVUS Biologicals, NB100-304) and PICH (ERCC6L; Proteintech, 15688-1-AP) were used for immunofluorescence at 1:1,000.

Techniques: Staining, Fluorescence, Comparison