Culturing U2OS Cells for Plk1 and Bora Studies

Human osteosarcoma U2OS cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 6% FCS (Lonza), 2 mM l-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin. Cell lines expressing LAP-Plk1, AKAP-LAP-Plk1, H2B-LAP-Plk1, and GFP-Bora under the control of tetracycline-inducible were cultured in DMEM containing Tet system approved fetal bovine serum (Lonza). Antibodies that were used were directed against Plk1 (18 (link), 19 (link)), Plk1, Cyclin B1, Actin (all from Santa Cruz), GFP (Roche), Plk1-pT210 (BD), Tubulin (Sigma), Bora (17 (link)), Aurora A (Cell Signaling) Histone H3-pS10, and H2AX (both from Upstate). The following drugs were used: BI 2536 (100 nM, Boehringer Ingelheim Pharma), MLN8054 (1 μM, Millennium Pharmaceuticals), thymidine (2.5 mM, Sigma), caffeine (5 mM, Sigma), adriamycin (0.5 μM, Sigma), nocodazole (250 ng/ml, Sigma), PI (Sigma) puromycin (Sigma, 2 μg/ml), and tetracyclin (Sigma, 1 μg/ml).

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Bruinsma W., Aprelia M., Kool J., Macurek L., Lindqvist A, & Medema R.H. (2015). Spatial Separation of Plk1 Phosphorylation and Activity. Frontiers in Oncology, 5, 132.

Publication 2015

Dulbecco’s modified FCS DMEM Cyclin B1 Plk1-pT210 Aurora A BI 2536 thymidine caffeine adriamycin nocodazole tetracyclin

Actin Adriamycin Akap Antibodies Aurora a Bi 2536 Caffeine Cell lines Cells Cyclin b1 Eagle Fetal bovine serum Glutamine Histone h3 Human Mln8054 Nocodazole Osteosarcoma Penicillin Pharmaceuticals Plk1 Puromycin Streptomycin Tetracyclin Thymidine Tubulin

Corresponding Organization :

Other organizations : Utrecht University

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Variable analysis

independent variables

Cell lines expressing LAP-Plk1, AKAP-LAP-Plk1, H2B-LAP-Plk1, and GFP-Bora under the control of tetracycline-inducible
BI 2536 (100 nM, Boehringer Ingelheim Pharma)
MLN8054 (1 μM, Millennium Pharmaceuticals)
Thymidine (2.5 mM, Sigma)
Caffeine (5 mM, Sigma)
Adriamycin (0.5 μM, Sigma)
Nocodazole (250 ng/ml, Sigma)
Puromycin (Sigma, 2 μg/ml)
Tetracyclin (Sigma, 1 μg/ml)

dependent variables

Plk1 (18, 19), Cyclin B1, Actin, GFP, Plk1-pT210, Tubulin, Bora (17), Aurora A, Histone H3-pS10, H2AX

control variables

Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 6% FCS (Lonza), 2 mM l-glutamine, 100 U/ml penicillin, and 100 mg/ml streptomycin
DMEM containing Tet system approved fetal bovine serum (Lonza)

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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