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We uncovered interlocking mechanisms regulating the temporal proteolysis of the transcriptional repressor E2F8 in cycling cells including SCFCyclin F in G2, dephosphorylation of Cdk1 sites, and activation of APC/CCdh1, but not APC/CCdc20 during mitotic exit and G1. Differential stabilization under limited APC/C activity allows E2F8 to reaccumulate during late G1 and coregulate S-phase entry.

Cancer is the second leading cause of death globally. Matching proper treatment and dosage is crucial for a positive outcome. Any given drug may affect patients with similar tumors differently. Personalized medicine aims to address this issue. Unfortunately, most cancer samples cannot be expanded in culture, limiting conventional cell‐based testing. Herein, presented is a microfluidic device that combines a drug microarray with cell microscopy. The device can perform 512 experiments to test chemosensitivity and resistance to a drug array. MCF7 and 293T cells are cultured inside the device and their chemosensitivity and resistance to docetaxel, applied at various concentrations, are determined. Cell mortality is determined as a function of drug concentration and exposure time. It is found that both cell types form cluster morphology within the device, not evident in conventional tissue culture under similar conditions. Cells inside the clusters are less sensitive to drugs than dispersed cells. These findings support a heterogenous response of cancer cells to drugs. Then demonstrated is the principle of drug microarrays by testing cell response to four different drugs at four different concentrations. This approach may enable the personalization of treatment to the particular tumor and patient and may eventually improve final patient outcome.

19E2F8 is a transcriptional repressor that antagonizes the canonical cell cycle 20 transcription factor E2F1. Despite the importance of this atypical E2F family member in cell 21 cycle, apoptosis and cancer, we lack a complete description of the mechanisms that control 22 its dynamics. To address this question, we developed a complementary set of static and 23 dynamic cell-free systems of human origin, which recapitulate inter-mitotic and G1 phases, 24 and a full transition from pro-metaphase to G1. This revealed an interlocking molecular 25 switch controlling E2F8 degradation at mitotic exit, involving dephosphorylation of Cdk1 26 sites in E2F8 and the activation of APC/C Cdh1 , but not APC/C Cdc20 . Further, we revealed a 27 differential stability of E2F8, accounting for its accumulation in late G1 while APC/C Cdh1 is 28 still active and suggesting a key role for APC/C in controlling G1-S transcription. Finally, we 29 identified SCF-Cyclin F as the ubiquitin ligase controlling E2F8 in G2-phase. Altogether, our 30 data provide new insights into the regulation of E2F8 throughout the cell cycle, illuminating 31 an extensive coordination between phosphorylation, ubiquitination and transcription in 32 promoting orderly cell cycle progression. 33 34 35 36 37 38 39 40 41 42 Ran et al., 2008, Ouseph, Li et al., 2012). Despite being part of the 'repressive' branch of E2F 57proteins, E2F7 and E2F8 belong to the pro-proliferative gene network underlying cell 58 proliferation (Cohen, Vecsler et al., 2013). 59 E2F1, as well as E2F7 and E2F8, are regulated post-translationally via temporal 60 proteolysis. The anaphase-promoting complex/cyclosome (APC/C) is a multi-subunit cell 61 cycle ubiquitin ligase and core component of the cell cycle machinery (King, Peters et al., 62 1995, Sudakin, Ganoth et al., 1995. The APC/C uses two related co-activators termed Cdc20 63 and Cdh1, which bind substrates and recruit them to the APC/C for ubiquitination and 64 subsequent degradation (Kernan, Bonacci et al., 2018). We previously identified both E2F7 65 and E2F8 as targets of the Cdh1-bound form of APC/C (APC/C Cdh1 ) (Cohen et al., 2013). 66These findings, together with other supporting studies (Boekhout, Yuan et al., 2016), shifted 67 the model by which the E2F1-E2F7-E2F8 circuitry communicates with the cell cycle clock to 68 regulate the transition from the G1-phase of the cell cycle into S-phase. Nevertheless, the 69 exact inter-dynamics of E2F1-E2F7-E2F8 circuitry throughout G1 and the mechanism by 70 which they are achieved are not entirely resolved. No less obscure is the interplay between 71 E2F1 and atypical E2Fs during G2-phase and mitosis. Dissecting these complex signaling 72 circuits is important for understanding the decision making mechanisms at two critical 73 points in the life of a proliferating cell -commitment to DNA replication and division. 74Cell-free systems are known for their capacity to reproduce complex cellular 75 processes in vitro while maintaining a physiologically relevant context, bridging the gap 76...

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