Fig. 5: Analysis of cisplatin response in ORC1 mutated cells.
a IGV coverage track images showing the identified point mutations in ORC1 (c.C1721T, p.T574M). Orange/green bar shows the proportion of reads that shows reference base G (orange, bottom panel) compared to mutant base A (green) in T302 primary and PDX tumours. Grey bars show 100% of sequenced bases at that location as reference. Bottom dark/light blue lane shows aminoacids based on reference sequence. b Clonogenic survival in untreated and cisplatin-treated WT RPE hTERT TP53-/- cells and cells with a point mutations in ORC1 (C1721T) introduced using the CRISPR/Cas9 system. Graph shows representative results for three independent experiments. Values of each data point are normalized to untreated conditions of the same genotype and data are depicted as mean ± SEM. c Representative images of clonogenic survival after cisplatin treatment for RPE-hTERT TP53-/- cells non-targeted or carrying ORC1C1721T/C1721T. Images show cells left untreated and cells treated with 0.5, 1, 2 or 5 µM cisplatin for 11 days, stained with crystal violet. d Formation of RAD51 foci in untreated and cisplatin-treated RPE hTERT TP53-/- cells and cells with CRISPR/Cas9 induced point mutations in ORC1. Plot depicts the mean±SEM of RAD51 foci of three independent experiments. Significance was calculated using a two-tailed t-test. e Representative images of RAD51 focus formation after irradiation in wild-type cells or ORC1C1721T/C1721T cells. Cells were irradiated with 10 Grey, then stained for RAD51 foci after 3 h. Rad51 foci are depicted in green, DAPI in blue.
