The ability to survive mitosis in the presence of microtubule poisons differs significantly between human nontransformed (RPE-1) and cancer (U2OS, HeLa) cells.

Cell Culture and Drug Treatment

HeLa (cervical carcinoma), U2OS (osteosarcoma) and hTERT-RPE-1 (RPE-1; retinal pigment epithelial) cell lines were cultured as monolayers in DMEM supplemented with 10% fetal bovine serum (FBS). All cells were maintained in a humidified incubator at 37°C in a 5% CO 2 environment. Prior to experiments cells were subcultured onto 25 mm 2 glass coverslips and grown as above for at least 16 h. For filming, coverslip cultures were mounted in Rose chambers and recordings were made at 37°C as previously described [ Khodjakov and Rieder, 2006 ]. Before imaging, nocodazole or taxol (Calbiochem, La Jolla, CA) was added to the medium at the concentrations noted in the text. The general caspase inhibitor (20 μM), Q-VD-OPh (R&D Systems, Minneapolis, MN; Cat # OPH001), was added to the medium 1 h before adding nocodazole.

Live-Cell Microscopy

Time-lapse recordings of RPE-1, HeLa and U2OS cells were captured every 2 (untreated controls) to 10 (experimental) minutes at 37°C with a low magnification (10X 0.3NA) PlanFluor phase-contrast objective lens mounted on a Nikon Diaphot microscope equipped with a Spot Insight QE video camera and fast UniBlitz shutters (Vincent Associates, Rochester, NY). The hardware was driven by Image-Pro Plus 5.1.020 (Media Cybernetics, Silver Spring, MD). Image sequences were compiled with ImageJ 1.35c (NIH), and contrast-adjustment was conducted using Photoshop CS2 (Adobe Systems). In our graphs N refers to the total numbers of cells pooled from ≥2 separate long-term recordings obtained on different days, with the exception of those experiments using the pancaspase inhibitor, Q-VD-OPh, which were from single experiments. In some cases the number of cells noted in the lanes of our timing graph ( Fig. 2 ) is different from that of our survival graph ( Fig. 4 ) because clustering of adjacent cells sometimes prohibited determining an accurate duration of mitosis for a particular cell but not its ultimate fate.

Immunofluorescence

Microtubule staining was performed as previously described [ Brito and Rieder, 2006 ]. As primary and secondary antibodies we used mouse anti-α-tubulin (DM1α, Sigma) and AlexaFluor 594 (Molecular Probes), respectively. DNA was counterstained with Hoechst 33342 (Molecular Probes). After staining, cells were imaged as a Z-series (200 nm apart) on an Olympus IX70 inverted microscope equipped with a CM350 camera (Photometrics). The images were deblurred using the SoftWorx 2.5 deconvolution algorithm (Applied Precision, Issaquah, WA), and presented as maximal intensity projections.