Fig. 2: Transcription–replication conflict creates RS on ecDNAs.
From: Enhancing transcription–replication conflict targets ecDNA-positive cancers
a, Schematics depicting transcription–replication conflict and RS. b, RS score 1 computed in TCGA patients grouped by ecDNA amplification status (n: 232, 582). c, DNA-fibre assay combined with MYC FISH in COLO320DM and COLO320HSR cells. Replication fork (RF) progression rate was measured globally (middle) or at the MYC locus (right) (box whiskers indicate min. to max.; n: 348, 317, 143, 101). d, Replication protein A phosphorylation: pRPA2-S33 IF combined with EGFR or MYC DNA FISH to show higher RS on ecDNA (n: 370, 274, 939, 568, 209, 244). e, pRPA2-S33 IF combined with EGFR FISH with 5-ethynyl-2′-deoxyuridine (EdU) added for 30 min; nuclei were co-stained by DAPI. Left, representative images. Second left, proportion of pixels with colocalization within each pRPA2-S33 pixel intensity bin (shade indicates median ± 25% quantile range, n: 10, 6). Second right, colocalization foci number (n: 267, 194, 104, 75). Right, percentage of pRPA2-S33 colocalized with EGFR (n: 371, 269). Red dot indicates percent of genome taken up by amplicon as calculated by WGS counts. f, Comparison of RS in tumour cells with different ecDNA content grouped by total DNA FISH intensity (GBM39ec, n: 111, 148, 111; COLO320DM, n: 282, 375, 282; PC3: 63, 83, 63). g, Genome tracks highlighting two regions within the ecDNA interval in COLO320 cells treated with triptolide or vehicle. h,i, Triptolide (TPL) treatment decreased RS on ecDNA in COLO320 (h) and GBM39 (i) cells (COLO320DM/HSR cells, n: 354, 350, 269, 130, 185, 161; GBM39ec/HSR cells, n: 139, 191, 264, 222). Boxplots b–i indicate centre line, median; limits, 25–75 quartiles; whiskers, 1.5× interquartile range or as otherwise specified. d–f,h–i were presented as violin plot and boxplot. Violin plot outlines kernel probability density. P determined by two-sided Wilcoxon test except unpaired Kolmogorov–Smirnov test in Fig. 2c. Scale bar, 10 µm (d (top row), e (left), h, i (top)), 2 µm (d (bottom row), e (right), i (bottom)), 50 kb (g (left)), 200 kb (g (right)).
