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Detail description of synthesis and supporting data

Figure S1. In vitro profiling of SCH900776 and its selected analogs. Figure S2. Analogs of MU380 with the N-trifluoromethylpyrazol pharmacophore. Figure S3. Dot plots of flow cytometric analysis using γH2AX and Annexin V staining related to Figure 2D. Figure S4. Dose response curves of relative viability of the tested cell lines treated with CHK1 inhibitors at indicated concentrations in combination with HU in the concentration range shown on the x-axis (related to Figure 3A). Figure S5. Dynamics of cytotoxic effects of CHK1 inhibitors alone and in combination with HU. Figure S6. Dose response curves of relative viability of the tested cell lines treated with CHK1 inhibitors at indicated concentration in combination with GEM in concentration range shown on the x-axis (related to Figure 3B). Figure S7. Dose response curves of relative viability of the tested cell lines treated with CHK1 inhibitors at indicated concentration in combination with irradiation in the dose range shown on the x-axis. Figure S8. Western blot analysis of total CHK1 and p-CHK1 (S296) levels in a panel of selected cell lines after indicated treatments. Figure S9. Drug retention analysis. Figure S10. MU380 induces CHK1 pS345 in mice hair follicles. Figure S11. Bioluminescent signal of SHO mice bearing MiaPaCa2 luc xenografts treated with GEM, SCH900776, or MU380 alone or in indicated combinations. Weight of MiaPaCa2 luc tumor-bearing animals. Representative images of γH2AX staining in MiaPaCa2 luc tumor sections.

Detail description of Material and Methods

Supplementary Legends for Supplemetary Figure S1 - S11

Summary Centrioles account for centrosomes and cilia formation. Recently, a link between centrosomal components and human developmental disorders has been established. However, the exact mechanisms how centrosome abnormalities influence embryogenesis and cell fate are not understood. PLK4-STIL module represents a key element of centrosome duplication cycle. We analyzed consequences of inactivation of the module for early events of embryogenesis in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). We demonstrate that blocking of PLK4 or STIL functions leads to centrosome loss followed by both p53-dependent and -independent defects, including prolonged cell divisions, upregulation of p53, chromosome instability, and, importantly, reduction of pluripotency markers and induction of differentiation. We show that the observed loss of key stem cells properties is connected to alterations in mitotic timing and protein turnover. In sum, our data define a link between centrosome, its regulators, and the control of pluripotency and differentiation in PSCs., Graphical Abstract, Highlights • Blocking of PLK4-STIL module in hESCs/hiPSCs leads to: • Centrosome loss, prolonged and error-prone mitosis; • p53-dependent differentiation; • Reduction of pluripotency linked to altered protein turnover, Recently a link has been established between centrosome and developmental disorders, yet the mechanisms connecting centrosome and cell fate are not understood. Cajanek and colleagues analyzed consequences of centrosome loss using hESCs/hiPSCs. They demonstrated that PLK4/STIL inhibition-mediated centrosome removal leads to loss of key stem cell properties connected to alterations in protein turnover and mitotic timing, which triggers p53-dependent differentiation.

International audience; Embryonic stem (ES) cells are pluripotent cells widely used in cell therapy and tissue engineering. However, the broader clinical applications of ES cells are limited by their genomic instability and karyotypic abnormalities. Thus, understanding the mechanisms underlying ES cell karyotypic abnormalities is critical to optimizing their clinical use. In this study, we focused on proliferating human and mouse ES cells undergoing multipolar divisions. Specifically, we analyzed the frequency and outcomes of such divisions using a combination of time-lapse microscopy and cell tracking. This revealed that cells resulting from multipolar divisions were not only viable, but they also frequently underwent subsequent cell divisions. Our novel data also showed that in human and mouse ES cells, multipolar spindles allowed more robust escape from chromosome segregation control mechanisms than bipolar spindles. Considering the frequency of multipolar divisions in proliferating ES cells, it is conceivable that cell division errors underlie ES cell karyotypic instability.

Fanconi anemia (FA) is a rare genetic disorder associated with bone-marrow failure, genome instability and cancer predisposition. Recently, we and others have demonstrated dysfunctional mitochondria with morphological alterations in FA cells accompanied by high reactive oxygen species (ROS) levels. Mitochondrial morphology is regulated by continuous fusion and fission events and the misbalance between these two is often accompanied by autophagy. Here, we provide evidence of impaired autophagy in FA. We demonstrate that FA cells have increased number of autophagic (presumably mitophagic) events and accumulate dysfunctional mitochondria due to an impaired ability to degrade them. Moreover, mitochondrial fission accompanied by oxidative stress (OS) is a prerequisite condition for mitophagy in FA and blocking this pathway may release autophagic machinery to clear dysfunctional mitochondria.

Ovarian cancer is one of the most common malignancies in women and contributes greatly to cancer-related deaths. Tumor suppressor candidate 3 (TUSC3) is a putative tumor suppressor gene located at chromosomal region 8p22, which is often lost in epithelial cancers. Epigenetic silencing of TUSC3 has been associated with poor prognosis, and hypermethylation of its promoter provides an independent biomarker of overall and disease-free survival in ovarian cancer patients. TUSC3 is localized to the endoplasmic reticulum in an oligosaccharyl tranferase complex responsible for the N-glycosylation of proteins. However, the precise molecular role of TUSC3 in ovarian cancer remains unclear. In this study, we establish TUSC3 as a novel ovarian cancer tumor suppressor using a xenograft mouse model and demonstrate that loss of TUSC3 alters the molecular response to endoplasmic reticulum stress and induces hallmarks of the epithelial-to-mesenchymal transition in ovarian cancer cells. In summary, we have confirmed the tumor-suppressive function of TUSC3 and identified the possible mechanism driving TUSC3-deficient ovarian cancer cells toward a malignant phenotype.

Automated imaging screens are performed mostly on fixed and stained samples to simplify the workflow and increase throughput. Some processes, such as the movement of cells and organelles or measuring membrane integrity and potential, can be measured only in living cells. Developing such assays to screen large compound or RNAi collections is challenging in many respects. Here, we develop a live-cell high-content assay for tracking endocytic organelles in medium throughput. We evaluate the added value of measuring kinetic parameters compared with measuring static parameters solely. We screened 2000 compounds in U-2 OS cells expressing Lamp1-GFP to label late endosomes. All hits have phenotypes in both static and kinetic parameters. However, we show that the kinetic parameters enable better discrimination of the mechanisms of action. Most of the compounds cause a decrease of motility of endosomes, but we identify several compounds that increase endosomal motility. In summary, we show that kinetic data help to better discriminate phenotypes and thereby obtain more subtle phenotypic clustering.