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. 2016 May 30;213(6):1011–1028. doi: 10.1084/jem.20151183

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© 2016 Zhang et al.

This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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Figure 1. — FA-like phenotype. (A) MMC sensitivity of SV40-transformed fibroblasts from the patient, FA, and control. (B) G2/M arrest in EBV lymphoblastoid B cells from the patient, his father, an unrelated healthy control, and an FA patient. Cells were either untreated (orange) or treated with 20 ng/ml (blue) or 50 ng/ml (red) MMC for 48 h. (C) Ubiquitination of FANCD2 after MMC treatment (150 ng/ml for 48 h) of EBV B cells from the patient, his parents, an unrelated healthy control, and an FA patient. (A–C) The experiment was performed three times. (D) Increase of chromosomal aberrations in cells from the patient after MMC treatment. Representative metaphase from SAS50 fibroblasts treated with MMC (50 ng/ml for 24 h). (a) Examples of chromosomal aberrations found in metaphase from the patient’s fibroblasts. (b) Chromosomal breaks. (c–e) Radial chromosomes. The histogram shows the frequency of chromosomal aberrations (i.e., breaks, radial chromosomes, and total aberrations) found in metaphase spreads from patient fibroblasts compared with normal fibroblasts at similar passage. Data are mean ± SEM. n = 75 and 71 metaphases, respectively. **, P = 0.002; ***, P = 0.0003 for Student’s t tests. This experiment was performed two times. (E) Estimation of telomere (Tel.) length from whole blood cells by terminal restriction fragment analysis. This Southern blot has been performed once. (F) Representative image of the SA–β-gal staining (cyan) in patient fibroblasts and in AT fibroblasts to measure cellular senescence. Nuclei were stained with propidium iodide (IP; red). Quantification of the percentage of SA–β-gal–positive cells versus total cells determined at different cell culture passages (p) in the different fibroblast cultures (control, patient, and AT). The percentage of SA–β-gal–positive cells in patient’s cultures remains at a low level (<11.5% at p28) compared to that of control cultures (15% at passage p31), whereas AT cells, known to present a premature senescent phenotype, exhibited a strong percentage of SA–β-gal–positive cells at earlier passages (39% at p23). This experiment was performed two times. Bar, 10 µm.