Skip to main content
. 2022 Dec 7;11:e80207. doi: 10.7554/eLife.80207

Figure 4. Extrachromosomal DNA (ecDNA) do not colocalize with large foci of the transcriptional machinery.

(A) Representative maximum intensity projection image of nascent EGFR RNA FISH (red) in E26 cell nucleus,(blue=DNA). Scale bar = 5 μm. Associated Ripley’s K function for this nucleus showing observed K function (red), max/min/median (black) of 10,000 null samples with p=0.05 significance cut-off shown (empty black circle). (B) Ripley’s K function for E26 nuclei (n=11) after EGFR nascent RNA FISH showing number of nuclei with significant and non-significant clustering at each given radius. All p-values for Ripley’s K function calculated using Neyman-Pearson lemma with optimistic estimate p-value where required, and Benjamini-Hochberg procedure (BHP, FDR = 0.05). (C) Representative maximum intensity projection images of immunoFISH in neural stem cell (NSC), E26 and E28 cell lines: Immunofluorescence for RPB1 (green) and EGFR DNA FISH (red). Scale bar = 5 μm. (D) Spearman’s correlation between number of EGFR foci and number of RPB1 foci, p = 0.13, E26 and E28 cell line data combined. (E) Violin plot of distribution of mean shortest interprobe distance per nucleus between EGFR foci and PolII foci in NSC (n=7), E26 (n=8) and E28 (n=7) cell lines. (F) As for (E) but for shortest single distance in each nucleus. ns, not significant. Kruskall-Wallis test. Statistical data relevant for this figure are in Figure 4—source data 1.

Figure 4—source data 1. Statistical data for Figure 4 and Figure 4—figure supplement 1.
EcDNA-large RPB1 foci distances for neural stem cell (NSC), E26 and E28 cell lines. Statistical analysis of data for DNA-ImmunoFISH (i – Figure 4E and F), RNA-ImmunoFISH (ii – Figure 4—figure supplement 1E F) EcDNA-large RPB1 foci distance (μm) indicated = median values shown. (iii) Median number of EGFR RNA FISH signals for NSC, E26 and E28 cell lines. (iv) Median ecDNA-large POLR2G foci distances for E28 mCherry-POLR2G cell line (Figure 4—figure supplement 1I, J). n = number of nuclei. Kruskall-Wallis and Mann-Whitney tests performed with comparisons as indicated.

Figure 4.

Figure 4—figure supplement 1. Analysis of sites of EGFR nascent transcription relative to RNA polymerase II in GBM cell lines.

Figure 4—figure supplement 1.

(A) Number of nascent EGFR RNA foci per cell line, at least 25 nuclei of each cell line imaged. Statistical significance examined by Mann-Whitney test. ns = not significant, ** p<0.01, ****p<0.0001 and are detailed in Figure 4—source data 1. (B) Representative images of nascent EGFR RNA FISH (shown in greyscale) in neural stem cell (NSC), E26 and E28 cell lines. MIP, scale bar = 5 μm. (C) Representative images of RNA polymerase II (RPB1) foci (arrow heads) detected by immunofluorescence. Scale bar = 5 μm. (D) Representative images of E26 and E28 nascent RNA immunoFISH for EGFR (red) and RNA polymerase II (PolII) (Rpb1 – green), MIP, scale bar = 5 μm. (E) Mean shortest interprobe distance between EGFR RNA and PolII foci. (F) As for (E) but for shortest distance. Median and quartiles plotted. Dotted line denotes y = 200 nm. Statistical significance examined by Mann-Whitney. ns, not significant. Statistical data relevant for this figure are in Figure 4—source data 1. (G) Representative images of immunoFISH in the E28 mCherry-POL2RG cell line: Immunofluorescence for mCherry and EGFR DNA FISH. Scale bar = 5 μm. (H) Violin plot of mean shortest distance per nucleus between EGFR foci and foci detected by Pol2RG-mCherry fusion. Dotted line denotes y=200 nm. (I) As for (H) but for shortest single distance in each nucleus. n=14 nuclei.