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Fig. 3 | Genome Biology

Fig. 3

From: Single-cell lineage analysis reveals genetic and epigenetic interplay in glioblastoma drug resistance

Fig. 3

Analysis of clonal cultures identifies distinct but functionally parallel chromosomal amplifications in “jackpot” drug resistant lineages. a Schematic depicts isolation of single cells and derivation of clonal cultures from multiple dasatinib-treated replicates of Experiments #1 (day 70; left) and 2 (day 56; right). b Chart details characteristics of those clonal cultures isolated from dasatinib experiments that were analyzed by low-coverage whole-genome sequencing (WGS), compared to drug-naïve GSCs (top row). Clones that exhibited features consistent with induced drug persistence (epigenetic) in these experiments are shown in the top part of chart. Clones that exhibited features consistent with genetic (acquired or preexisting) resistance are shown in the bottom part of chart. For each single-cell-derived clone, the chart indicates the experiment and replicate from which it was derived (Clone ID), the DNA barcode identity (Lineage ID), and the rank and abundance of the respective lineage in the gDNA analysis of the corresponding experiment (day 70 for Experiment #1; day 56 for Experiment #2). Genomic tracks depict read density in low-coverage WGS over chromosomal bands 13q34 (containing IRS2) and 2q36 (containing IRS1). The final column summarizes the extent to which the clone loses dasatinib resistance after a drug-free washout period. c Single-cell-derived clones were subjected to a drug-free washout period and then re-challenged with dasatinib. Plot depicts growth in the presence of dasatinib, relative to drug-naïve GSC8 cells (Y-axis); all conditions were treated independently in at least 4 replicates. Circles represent the mean relative growth of individual clonal cultures. Horizontal black bars reflect the mean growth of clones from the indicated lineage (top). d Barplots depict relative growth of drug-naïve GSC8 cells transduced with IRS1, IRS2, or luciferase expression constructs (**, p < 0.01 by two-tailed Student’s t test; standard error bars depicted). Cells were grown at the indicated dasatinib concentrations. Western blot shows IRS1, IRS2, and Actin protein expression in the indicated GSC8 cultures. Overexpression of IRS1 or IRS2 confers dasatinib resistance

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