Supplemental Figure S1: Cytotoxicity of MK-1775 in BJ fibroblasts and neonatal rat myocardiocytes.

(A)Confluent or log-phase BJ fibroblasts were treated with different concentrations of MK-1775 in 6-well plates. After 4 days, cells were fixed in methanol and stained using crystal violet. (B) Primary neonatal rat cardiomyocytes were isolated and cultured in DMEM medium containing 10% FCS as previously described (Lu et al 2010, PLoS One, 5(9):e12963). After three days of culturing, cells were treated with MK-1775 (100 nM or 300 nM) and 24 hours later beating was analyzed using bright field live microscopy. Average beating frequencies (beatings per minute) and standard deviations of 4 independent cultures are indicated (see for representative examples Suppl. Movies M1, M2 and M3). (C) Neonatal rat myocardiocytes grown on laminin-coated glass coverslips were treated as for panel B. Cells were fixed in formaldehyde and stained for Troponin T/Alexa-488, -H2AX/Alexa-568 and counterstained with Hoechst (Sigma). Representative images are shown. Arrow indicates a co-isolated fibroblast without troponin T bundles showing MK-1775-induced -H2AX foci. (D) Primary neonatal rat cardiomyocytes were treated as for panel C. Numbers of -H2AX foci per nucleus were counted and averages and standard deviations of three experiments (at least 25 nuclei per condition quantified) are shown.

Supplemental Figure S2: Cytotoxicity andradiosensitization of MK-1775 in cancer cells.

(A) MTT assays of MCF-7, MDA-MB-231, SK-BR-3, T47D and HeLa cells were performed as for Fig. 1(I). For each cell line, 2000 cells were plated (6 replicates) and MTT conversion was measured after 4 days of treatment. Untreated cells were set to 100%. Survival curves and IC50 values were determined using Graphpad software. (B) MK-1775 concentrations at which 50% growth inhibition is observed (IC50) are indicated for indicated cell lines. (C) Long term clonogenic survival assays with MDA-MB-231 and SK-BR-3, T47D and Hela were performed as described in Fig. 1(J,K). ‘*’indicates p<0.05,‘**’indicates p<0.01, ‘***’indicates p<0.001.

Supplemental Figure S3: Forced activation of Cdk1 impairs homologous recombination DNA repair.

(A)MCF7 cells were treated with PD-166285 (500nM) for indicated time-points and analyzed by Western blotting.(B)HeLa-pDR-GFP cells (kindly provided by Dr. J. Parvin, Ohio State University) were treated with MK-1775(300nM) or PD-166285 (500nM) at 1 hour before transfection withI-Sce1.After 48 hours, at least 100.000 events were analyzed per sample for GFP positivity. (C)HeLa-pDR-GFP cells were co-transfected with I-Sce1 and pSuper-Wee1 or were treated with MK-1775(300nM) at 1 hour before transfection and analyzed as for panel 3F. (D)HeLa-pDR-GFP cells were transfected with I-Sce1 in combination with pECFP (Clontech), pECFP-wt-Cdk1 or pECFP-AF-Cdk1. If indicated, cells were treated with MK-1775 (300nM) at 1 hour before transfection. After 48 hours, at least 100.000 events were analyzed per sample. Representative plots are shown. The amounts of GFP-positivity in CFP positive cells were quantified from three independent experiments. Averages and standard deviations are shown.‘*’indicates p<0.05,‘**’indicates p<0.01.

Supplemental Figure S4: Mitotic entry after Wee1 or Cdk1 inhibition.

MCF-7 cells were treated as for Fig. 4C. After treatment, cells were harvested and stained for phospho-S3291-BRCA2/Alexa-647 and p-H3/Alexa-488 cells and analyzed by flow cytometry. Graphs show pH3/Alexa-488 positivity.A minimum of 10.000 events per sample was analyzed and averages and standard deviations of three experiments are shown.

Supplemental Movie M1: bright field live microscopy of untreated primary neonatal rat cardiomyocytes.

Supplemental Movie M2:bright field live microscopy of primary neonatal rat cardiomyocytes, after 24h of treatment with MK-1775 (100nM).

Supplemental Movie M3:bright field live microscopy of primary neonatal rat cardiomyocytes, after 24h of treatment withMK-1775 (300nM).

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