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. 2022 Dec 7;11:e80207. doi: 10.7554/eLife.80207

Figure 1. The nuclear localization of extrachromosomal DNA (ecDNA) in glioblastoma (GBM) cell lines.

(A) Whole genome sequencing (WGS) and AmpliconArchitect analysis for ecDNA regions for E26 and E28 cell lines showing an EGFR exon 2–7 deletion in all ecDNA in E26 cells (seen in WGS and AmpliconArchitect regions a and b), and a subpopulation of ecDNA in E28 with a deletion across EGFR exons 7–14 (seen in WGS and Amplicon Architect region a – no deletion in E28 AmpliconArchitect region b). Genome coordinates (bp) are from the hg38 assembly of the human genome. (B) DNA FISH on metaphase spread of the E26 cell line showing EGFR (green) present on ecDNA, and on a homogeneously staining region (HSR) (arrowed) detected in ~10% of metaphases. Scale bar = 10 μm. (C) As for (B) but for the E28 cell line. (D) Violin plot of the number of EGFR DNA FISH signals per metaphase spread of E26 and E28 cells. Median and quartiles are shown. ** p=0.008 (Mann-Whitney test). Median values are 51 (E26)and 12 (E28), n=25 (E26) and 24 (E28) spreads. (E and F) Representative DNA FISH images of metaphase spread (E) and 2D nuclei (F) for neural stem cell (NSC), E26, and E28 cells showing signals for chromosome 7 (red) and EGFR (green). Blue = DNA (DAPI). Scale bar = 10 μm. The five erosions bins from the periphery to the centre of the nucleus are shown in F. (G) EGFR FISH signal intensity normalized to that for chromosome 7 (EGFR:Chr7 Mean Intensity) across five bins of equal area eroded from the peripheral (Bin 1) to the centre (Bin 5) of the nucleus for NSC, E26, and E28 cell lines. Median and quartiles shown. **** p<0.0001, * p<0.05. Kruskall-Wallis test. EGFR and chr7 signal normalized to DAPI shown in Figure 1—figure supplement 1. n=66 (NSC), 59 (E26), 64 (E28) nuclei. Statistical data relevant for this figure are in Figure 1—source data 1.

Figure 1—source data 1. Statistical data for Figure 1 and Figure 1—figure supplement 1.
Median values for number of EGFR DNA FISH signals per metaphase spread for E26 and E28 cell lines. Data are for Figure 1D. Mean Chr7 (Texas Red) and EGFR (FITC) DNA FISH signal intensity in bins eroded from the periphery (1) to the centre (5) of the nucleus of neural stem cell (NSC), E26 and E28 cells. p-Values from Kruskall-Wallis test. Data are for Figure 1G and Figure 1—figure supplement 1.

Figure 1.

Figure 1—figure supplement 1. Additional EGFR and chromosome 7 signal intensity data.

Figure 1—figure supplement 1.

Radial distribution, normalized to DAPI, across bins of equal area eroded from the edge (1) to the centre (5) of the nucleus for (A) chromosome 7 (TxR mean normalized intensity per nucleus) or (B) EGFR (FITC mean normalized intensity per nucleus) hybridization signals.
Median and quartiles are shown. Statistical significance was examined by Kruskall-Wallis. **** p<0.0001. Statistical data relevant for this figure are in Figure 1—source data 1.