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. 2021 Jan 25;11:2157. doi: 10.1038/s41598-021-81075-0

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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BLM-deficient cells exhibit high levels of ROS, oxidative DNA damage, and ROS-dependent reduction of DNA replication speed. (A) Flow cytometry measurement of cellular superoxide levels in GM00637 (BLM^+/+) and KSVS1452 (BLM^KO) cells stained with the super-oxide specific probe dihydroethidium (DHE). Analysis was performed in triplicate and mean ± SD is shown. (B) Fluorescence measurements of 2′,7′-dichlorofluorescin diacetate (DCFA)-stained BLM-proficient GM00637 (BLM^+/+) cells, Bloom-syndrome-patient-derived GM08505 (BLM^−/−) cells, BLM-knockout cells KSVS1452 (BLM^KO) and BLM-complemented cells KSVS1454, indicating cellular ROS. Analysis was performed in triplicate and mean ± SD is shown. (C) Flow cytometry measurements of MitoSOX-stained (mitochondrial ROS) BLM-proficient GM00637 (BLM^+/+) cells, Bloom-syndrome-patient-derived GM08505 (BLM^−/−) cells, BLM-knockout cell line KSVS1452 (BLM^KO) and BLM-complemented cell line KSVS1454. Analysis was performed in triplicate and mean ± SD is shown. (D) Representative fluorescence microscopy images of live GM00637 (BLM^+/+), GM08505 (BLM^−/−) and KSVS1452 (BLM^KO) cells stained with MitoSOX (red foci), indicating mitochondrial ROS, and with Hoechst 33342 (blue). Scale bars 10 µm. (E) Confocal microscopy images of fixed GM00637 (BLM^+/+), GM08505 (BLM^−/−) and KSVS1452 (BLM^KO) cells immunostained for oxidative damage to DNA (antibody 8-oxo-dG, 15A3, SCBT). GM00637 (BLM^+/+) cells exposed to 100 µM H₂O₂ for 2 h served as a positive control for oxidative nucleotide damage. Scale bars 10 µm. (F) Median fluorescence intensity was determined by flow cytometry in fixed GM00637 (BLM^+/+), GM08505 (BLM^−/−) and KSVS1452 (BLM^KO) cells immunostained with antibody 8-oxo-dG 15A3 (SCBT). Analysis was performed in triplicate and mean ± SD is shown. (G) Fluorescence measurements of 2′,7′-dichlorofluorescin diacetate (DCFA)-stained BLM-proficient GM00637 (BLM^+/+) cells and BLM-knockout cells KSVS1452 (BLM^KO) either treated with 5 mM antioxidant N-acetyl-cysteine (NAC) for 24 h, or untreated. Analysis was performed in triplicate and mean ± SD is shown. (H) Measurement of DNA replication speed in GM00637 (BLM^+/+) cells and BLM-knockout cells KSVS1452 (BLM^KO) either treated with 5 mM antioxidant N-acetyl-cysteine (NAC) for 24 h, or untreated, prior to DNA fiber analysis. Significance of differences was determined by a Student’s t-test and is reported as *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001; ns, not significant. Flow cytometry data were analyzed with FlowJo v. 10.7 software (BD Life Sciences, https://www.flowjo.com/solutions/flowjo/downloads).