Fig 2: PLK1 inhibition induces a sustained change in cell fate.A Schematic of the drug wash-out experiment. SUM149PT cells were treated for 3 days with rigosertib or DMSO. Subsequently, the drug was washed out and cells were cultured for eight more days without the drug and then harvested for downstream experiments. B Immunoblot showing levels of ERa and ERK2 (loading control) in SUM149PT cells treated with the indicated concentrations of rigosertib as depicted in Fig. 4A. C Bar graphs representing average mRNA expression of ESR1 and downstream targets in SUM149PT cells treated with 100 nM rigosertib or DMSO as depicted in Fig. 4A. n = 2 experimental replicates with 2 technical replicates each. Unpaired Student’s t-test. Data are means ± SD. D Representative flow-cytometry dot plots of EdU/Hoechst cell cycle staining of SUM149PT cells treated with 100 nM rigosertib or DMSO as depicted in Fig. 4A. E Bar graph depicting the proportion of cells in different cell cycle states based on EdU/Hoechst cell cycle staining shown in Fig. 4D. n = 3 experimental replicates. Ordinary two-way ANOVA with multiple comparisons. Data are means ± SD. F Schematic of the experimental setup for in vitro treated SUM149PT cells grown as mouse xenografts in NSG mice. Cells were treated for 3 days with 1 µM rigosertib or DMSO prior to injection. G Kinetics of primary tumour growth of SUM149PT cells treated in vitro with 1 µM rigosertib (n = 5 mice) or DMSO (n = 5 mice) as depicted in Fig. 4F. Mann–Whitney U-test. Data are means ± SD. H Left panel: Pie charts depicting quantification of tumour incidence upon in vitro treatment of SUM149PT cells as in Fig. 4F. Right panel: Table summarizing the frequency of tumour initiating cells (TICs) and respective statistical analysis. Chi-square test.

Fig 3: High-throughput drug screen reveals estrogen receptor a (ERa) induction in triple-negative breast cancer (TNBC) upon polo-like kinase 1 (PLK1) inhibition.A Schematic of the high-throughput drug screen to identify inhibitors that induce ERa signalling in TNBC. Cells without active ERa signalling do not express GFP, whereas cells with active ERa signalling trigger the ERE-GFP reporter and express GFP. Compounds were added for 48 h to SUM149PT ERE-GFP cells. GFP signal and Hoechst signal were measured with fluorescence microscopy in living cells. B Dot plot depicting GFP signal and nuclei number derived from Hoechst staining from the primary drug screen. C Dot plot depicting GFP signal and nuclei number derived from Hoechst staining from the secondary validation screen. Each point represents the mean of three technical replicates. Hits were classified as proliferative, cytostatic or toxic. PLK1 inhibitors are depicted in red. D Representative fluorescence microscopy live-cell images from the validation screen shown in Fig. 1C. SUM149PT cells were treated with the indicated PLK1 inhibitors for 48 h. The ERE-GFP signal is depicted in green, the Hoechst nuclei stain in blue. Scale bars: 100 µm. E Immunoblot showing levels of ERa and ERK2 (loading control) in SUM149PT cells treated for 72 h with rigosertib or DMSO at the indicated concentrations. F Bar graph representing average mRNA expression of ESR1 in SUM149PT cells treated for 72 h with rigosertib or DMSO. n = 2–3 experimental replicates with 2 technical replicates each. Ordinary one-way ANOVA with multiple comparisons. Data are means ± SD. G Bar graph representing flow-cytometry analysis of ERE-GFP positive cells after rigosertib treatment or transfection with indicated siRNA for 72 h. n = 6 experimental replicates. Kruskal-Wallis test. Data are means ± SD. H Heatmap depicting early estrogen response proteins (from Molecular Signatures Database [MSigDB] hallmark gene sets) changing significantly upon rigosertib treatment (n = 2 experimental replicates) compared to DMSO (n = 3 experimental replicates). Data is row-normalized. I Dot plot showing PLK1 expression in ERa positive versus ERa negative breast cancer samples in the METABRIC [26, 27] cohort. Unpaired Student’s t-test. J Dot plot depicting PLK1 expression in different breast cancer subtypes in the METABRIC [26, 27] cohort. Ordinary one-way ANOVA with multiple comparisons. K Dot plot depicting PLK1 expression in different breast cancer cell lines from the Cancer Cell Line Encyclopedia (CCLE) [82]. Unpaired Student’s t-test.

Fig 4: Microvascular endothelial expression of estrogen receptors. Estrogen receptor-ß (red, ER-ß, Thermo Fisher, MA5-13304; green, CD31, R&D systems, AF3628; blue, DAPI) expression in untreated arterioles from females <40 yr (n = 3; A) and =40 yr (n = 4; B), and biological males (n = 3; C). D: mean intensity of ER-ß normalized to endothelial area identified using CD31 staining. G protein-coupled estrogen receptor (red; Thermo Fisher, PA5-77396) costained with CD31 (green; R&D systems, AF3628) and DAPI (blue) in arterioles from females <40 (n = 3; E) and =40 (n = 4; F) yr of age, as well as biological males (n = 3; G). H: mean intensity of GPER-signal normalized to endothelial area identified using CD31 staining. Top, right: for visualization purposes only, magnified sections of each arteriole with a 40% increase in both the brightness and contrast. Representative immunohistochemical staining for estrogen receptor-a (Thermo Fisher, MA5-14501) in females <40 (I) and =40 (J) yr of age, as well as biological males (K). *P < 0.05: one-way ANOVA with Fisher’s least significant difference (LSD) test (D and H). GPER, G protein-coupled estrogen receptor; ER-ß, estrogen receptor-ß.