To examine the cellular efficacy of Plk1-PBD inhibitors generated in this study, MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt]-based assays were performed using Cell Titer 96 AQueous One Solution Cell Proliferation Assay kit (Promega, WI) according to the manufacturer’s instructions. In brief, asynchronously growing HeLa cells were plated at a density of 2000 cells/100 μL medium in a 96-well plate in triplicate. The cells were treated with compounds at the indicated concentration for 24 h followed by another 24 h treatment after replacing them with fresh medium (a total of continuous 48 h treatment). The resulting cells were treated with 20 μL of the MTS solution, incubated at 37 °C for 1–4 h in a humidified 5% CO2 atmosphere, and subjected to absorbance measurement at 490 nm using the Perkin-Elmer EnSpire Multimode 96-well plate reader (Waltham, MA).
Enspire multimode 96 well plate reader
Manufactured by PerkinElmer
The EnSpire Multimode 96-well plate reader is a compact, high-performance device designed for a variety of assays in life science research. It is capable of absorbance, fluorescence, and luminescence detection in 96-well microplates.
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3 protocols using enspire multimode 96 well plate reader
1
Cellular Efficacy of Plk1-PBD Inhibitors
2
Cell Viability Assay Protocol
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Cell viability was determined using the calorimetric CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) kit (Promega) containing a tetrazolium compound [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS)] and an electron-coupling phenazine ethosulfate. For the MTS assays in Fig. 1B , L363 cells cultured in a 96-well plate were treated with the indicated compounds for 48 h (the final volume of 100 μL). After the compound treatment, cells were additionally treated with 20 μL of the MTS solution and incubated at 37 °C for 2 to 4 h in a humidified 5% CO2 atmosphere. The absorbance of the resulting sample was measured at 490 nm using a PerkinElmer EnSpire Multimode 96-well plate reader. For the MTS assays in Fig. 4E , U2OS cells stably expressing either Pendo-mGFP-Plk1 WT or F559D were seeded at approximately 5,000 cells/well in a 96-well plate, depleted of endogenous Plk1 by RNAi, and then treated with either control DMSO or Allopole as indicated in the schematic diagram in SI Appendix, Fig. S4E .
3
ROS Measurement in TGF-β1 and T0901317 Treated Hep3B Cells
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Hep3B cells were seeded at a density of 4 × 104 cells in 12-well dishes, serum-starved for 16 h, and then, at 80% confluency, cells were treated with TGFβ1 (5 ng/ml) or T0901317 (5 μM), or a combination thereof for 16 h. Where indicated, Hep3B cells were transiently transfected with the indicated siRNAs, as described above. Afterwards, cells were seeded at the indicated concentration, serum-starved, and treated as described. After treatment, cells were incubated with 2.5 μM 2″,7′-dichlorodihydrofluorescein diacetate (Invitrogen, Thermofisher Scientific) in HBSS without red phenol for 30 min. Cells were subsequently lysed with a buffer containing 25 mM HEPES (pH 7.5), 60 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, and 0.1% Triton X-100 for 10 min at 4 °C. Fluorescence analysis was performed in an Enspire Multimode 96-well plate reader (Perkin Elmer). Fluorescence values were normalized relative to the protein content and are presented as percentage of control. Average of four independent biological replicates in technical duplicate is presented.
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