Fig. 1: Breast cancer cells show aberrant 5mC oxidation and increased LINE 1 ORF1p level. | Nature Communications

Fig. 1: Breast cancer cells show aberrant 5mC oxidation and increased LINE 1 ORF1p level.

From: Isoform-specific and ubiquitination dependent recruitment of Tet1 to replicating heterochromatin modulates methylcytosine oxidation

Fig. 1

A Gene structure of the human TET1 locus. Non-coding exons 1 and 3 harbor different transcription start sites, targeted by a different promoter. Translation starts in exon 2 (TET1) and 4 (TET1s). TET1 protein functional domains are indicated. Graphical representation of cytosine modifications and their maintenance during the cell cycle. B Fiji-based in situ cytosine modifications analysis procedure. MCF cells were immunostained and imaged using confocal microscopy. Binary nuclear and heterochromatin masks were generated based on DNA signals (DAPI). Mean fluorescence intensities in the respective areas were measured. C Boxplots showing normalized mean intensity of cytosine modifications at heterochromatin regions. n (5mC) = 23 (MCF10a) - 17 (MCF7), n (5hmC) = 19 (MCF10a) - 20 (MCF7), n (5fC) = 21 (MCF10a) - 17 (MCF7), n (5caC) = 17 (MCF10a) - 16 (MCF7) cells. D, E Live-cell analysis of Tet1 and Tet1s subnuclear localization: confocal images of MCF10a and MCF7 cells expressing EGFP-Tet1s/EGFP-Tet1 and mRFP-PCNA 8 h post-transfection. Colocalization of Tet1-X with PCNA was examined by line-profile analysis and relative protein accumulation in early and late S-phase. n (TET1) = 14 (MCF10a) - 13 (MCF7), n (Tet1s) = 16 (MCF10a) - 17 (MCF7) cells. F Synchronized MCF cells immunostained against LINE 1 ORF1p. Cytoplasmic fluorescence mean intensity levels were plotted. Representative confocal images for these immunostainings, including endogenous TET1/TET1s, are shown in G. For all boxplots, the box represents 50% of the data, starting in the first quartile (25%) and ending in the third (75%). The line inside represents the median. The whiskers represent the upper and lower quartile. Statistical significance was tested with a paired two-samples Wilcoxon test (n.s. not significant, is given for p-values ≥ 0.05; one star (*) for p-values < 0.05 and ≥ 0.005; two stars (**) is given for values < 0.005 and ≥ 0.0005; three stars (***) is given for values < 0.0005). N-numbers and p-values are shown in Supplementary Data 1. Source data are provided as a Source Data file. Scale bars = 5 µm.

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