Mitotic Kinase Inhibition and Proteasome Blockade

Mitotic enriched extracts were obtained from parental HeLa cells incubated for 12-16 h in media containing 5 μM of STLC (S-trityl-L-cysteine, Sigma) before harvesting by mitotic shake off. For inhibition of different mitotic kinases the following drugs were used for 1 h at 37ºC: 10 μM RO 3306 (Cdk1 inhibitor, Enzo Life Sciences), 100 nM BI 2536 (Plk1 inhibitor, Axon Medchem), 2 μM ZM 447439 (AurkB inhibitor, AstraZeneca), 5 μM MPS1-IN-1 (Kwiatkowski et al. 2010 (link)) and 40 mM LiCl (GSK3β inhibition). Mitotic exit was blocked with 10 μM of the proteasome inhibitor MG132 (EMD Millipore).

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Ferreira L.T., Logarinho E., Macedo J.C., Maia A.R, & Maiato H. (2021). SOGA1 and SOGA2/MTCL1 are CLASP-interacting proteins required for faithful chromosome segregation in human cells. Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology, 29(2), 159-173.

Publication 2021

S-trityl-L-cysteine RO 3306 BI 2536 ZM 447439 MG132

Axon Bi 2536 Cdk1 Drugs Gsk3β Hela cells Inhibition Kinases L cysteine Mg132 Mps1 Parental Plk1 Proteasome inhibitor Ro 3306 Shake Zm 447439

Corresponding Organization :

Other organizations : i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Universidade do Porto

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Variable analysis

independent variables

5 μM of STLC (S-trityl-L-cysteine, Sigma)
10 μM RO 3306 (Cdk1 inhibitor, Enzo Life Sciences)
100 nM BI 2536 (Plk1 inhibitor, Axon Medchem)
2 μM ZM 447439 (AurkB inhibitor, AstraZeneca)
5 μM MPS1-IN-1 (Kwiatkowski et al. 2010)
40 mM LiCl (GSK3β inhibition)

dependent variables

Mitotic exit

control variables

Parental HeLa cells
Incubation for 12-16 h in media
Harvesting by mitotic shake off
Mitotic exit blocked with 10 μM of the proteasome inhibitor MG132 (EMD Millipore)

controls

Positive control: Mitotic exit blocked with 10 μM of the proteasome inhibitor MG132
Negative control: Not explicitly mentioned

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