Figure 3: Transcriptional, cellular and karyotypic characterization of SACi in aneuploid cells.

(a) Schematics of gene expression profiling. HCT116 and HPT cells were treated with two SAC inhibitors, reversine (250nM and 500nM) and MPI-0479605 (250nM), global gene expression profiles were generated at 6hr, 24hr and 72hr post-drug exposure, and gene set enrichment analysis was performed to compare the transcriptional effect of SACi. (b) Unsupervised hierarchical clustering of the 4 cell lines based on drug-induced transcriptional changes. (c) Time from mitotic arrest to division following nocodazole (200ng/mL) treatment. **, p<0.01; two-tailed t-test. Bar, median; box, 25^th and 75^th percentile; whiskers, 1.5 X interquartile range; circles, individual cells. (d) Flow cytometry-based quantification of G2/M phase arrest induced by 48hr exposure to MPI-0479605 (250nM). ****, p=1e-05; two-tailed t-test. (e) The prevalence of micronuclei formation in HCT116 and HPT cells cultured under standard conditions or exposed to reversine (500nM) for 24hr. *, p=0.007; ***, p<0.001; two-tailed Fisher’s exact test. (f) Representative images of micronuclei formation in HPT2 cells exposed to reversine (500nM) for 24hr. Scale bar, 10μm. (g) Flow cytometry-based quantification of sub-G1 cell fraction induced by 48hr (left) or 14d (+3d of recovery; right) exposure to MPI-0479605 (250nM). **, p=0.002; ****, p=3e-05; two-tailed t-test. (h) Genome-wide copy number profiles of HCT116 and HPT single cells, as generated by the AneuFinder algorithm, at baseline (untreated; do), after 3d of MPI-0479605 (250nM) treatment (ongoing SACi; d3), and after recovery from 14d of treatment (recovered; d14+3). Individual cells are represented as rows, with chromosomes plotted as columns. In all plots, data represent the mean ± s.d. unless otherwise noted; n=3 biological replicates in all experiments unless otherwise noted.