Journal: Cell death discovery

Article Title: Polyploidy mitigates the impact of DNA damage while simultaneously bearing its burden.

doi: 10.1038/s41420-024-02206-w

Figure Lengend Snippet: Fig. 2 DNA damage accumulation in diploid and polyploid cells. A–D RPE1-Fucci cells were treated with DCB (10 μM) for 2 days. Microscopic images and histograms indicating the DNA content of G1 phase cells are shown in A. Neutral comet assay, representative immunofluorescence images, and the number of γH2AX- or 53BP1-positive foci per cell (n = 155 in control and 71 in DCB) are shown in B, C, and D, respectively. E–G Analysis of DNA damage accumulation in sorted Huh7-Fucci cells. G1 diploid, G1 polyploid, and S/G2/M cells were sorted using FACS from Huh7-Fucci cells cultured under normal conditions. Immunofluorescence images, the number of 53BP1-positive foci per cell (n = 50), and olive tail moments in neutral comet assay are shown in E, F, and G, respectively. In all dot plots, data were obtained from three or more independent experiments. Error bars represent the standard deviations of the mean. *p < 0.05, **p < 0.01. Student’s t-test. Scale bars, 50 μm in A and 10 μm in C, E.

Article Snippet: CTR CDDP DXR R el at iv e ex pr es si on **# * **** ** D E GF Diploid Polyploid1EPR7huH NES = -2.00 FDR < 0.01 Polyploid Diploid Mitotic spindle DNA damage/ telomere stressinduced senescence Senescence-associated secretary phenotype Polyploid Diploid Polyploid Diploid NES = -1.94 FDR < 0.01 NES = 2.10 FDR < 0.01 Log2 Fold Change (polyploid/diploid) -lo g 1 0 (F D R ) Sorting DNA damage evaluation RNA sequencing qRT-PCR Huh7-Fucci cells G1 Diploid Polyploid A za le aB 5 DNA amounth2-3 H H um an IL -6 ( pg /m l) CTR CDDP DXR *** ** Huh7 Diploid Polyploid Cell Death Discovery (2024) 10:436 (Addgene plasmid # 57384) [36] and 3xnls-mNeonGreen (Addgene plasmid # 98875) [37] and was subcloned into a sleeping-beauty transposon plasmid harboring a puromycin selection cassette.

Techniques: Neutral Comet Assay, Control, Cell Culture

Journal: Cell death discovery

Article Title: Polyploidy mitigates the impact of DNA damage while simultaneously bearing its burden.

doi: 10.1038/s41420-024-02206-w

Figure Lengend Snippet: Fig. 3 Comparison of DNA damage accumulation in diploid and stable polyploid Huh7 cells. A Representative histograms of the DNA content. B Metaphase chromosomes stained with Giemsa. C Olive tail moment in the neutral comet assay. n = 96–191 cells per group. D, E Immunofluorescence images of 53BP1 and γH2AX staining. F, G The number of γH2AX- or 53BP1-positive foci per cell. n = 50–52 cells per group. Diploid and stably polyploid Huh7-αTubulin-mScarlet cells were used in C–G. In C, F, and G, data were obtained from three or more independent experiments, and error bars represent the standard deviations of the mean. The number of genomic (H) and mitochondrial (I) DNA damage lesions evaluated by LORD-Q. Three independent diploid and stable polyploid cell lines established from Huh7 cells were analyzed in H, I. Each dot represents one cell line in H and I, and the variation in each cell line is shown in Fig. S5. Each cell line was analyzed in three independent experiments. The cells were treated with or without cisplatin and doxorubicin for a specified duration. CDDP cisplatin, DXR doxorubicin. *p < 0.05, **p < 0.01. Student’s t-test. Scale bar, 50 μm.

Article Snippet: CTR CDDP DXR R el at iv e ex pr es si on **# * **** ** D E GF Diploid Polyploid1EPR7huH NES = -2.00 FDR < 0.01 Polyploid Diploid Mitotic spindle DNA damage/ telomere stressinduced senescence Senescence-associated secretary phenotype Polyploid Diploid Polyploid Diploid NES = -1.94 FDR < 0.01 NES = 2.10 FDR < 0.01 Log2 Fold Change (polyploid/diploid) -lo g 1 0 (F D R ) Sorting DNA damage evaluation RNA sequencing qRT-PCR Huh7-Fucci cells G1 Diploid Polyploid A za le aB 5 DNA amounth2-3 H H um an IL -6 ( pg /m l) CTR CDDP DXR *** ** Huh7 Diploid Polyploid Cell Death Discovery (2024) 10:436 (Addgene plasmid # 57384) [36] and 3xnls-mNeonGreen (Addgene plasmid # 98875) [37] and was subcloned into a sleeping-beauty transposon plasmid harboring a puromycin selection cassette.

Techniques: Comparison, Staining, Neutral Comet Assay, Stable Transfection