Glass bottom coverslip dishes

Cells were plated on glass-bottom coverslip dishes (MatTek, AshLand, MA, USA) the day prior to imaging experiments. Cells were washed once with PBS and then incubated with 70 nM of MitoTracker Green FM (Life Technologies, CA, USA) for 20 min at 37°C. Cells were washed twice with phenol red-free medium. All live cell imaging experiments were performed in a live cell imaging chamber in the presence of 5% CO₂ at 37°C using the confocal laser microscope LSM 700 (Zeiss Canada, North York, ON, Canada). The LSM 700 is equipped with the PC running LSM ZEN 2012 (Version 8) software. Mitochondria were visualized using laser 488 power (3-6%) and the plan-Apochromat 40×/1.4 or 63×/1.4 NA oil immersion objectives. At least ten images, z-stacks or time-lapse images were obtained for each cell line. The Fiji software [56 (link)] was used to adjust the intensity, split frames and crop images. Mitochondrial length was measured as described previously [57 (link)]. For quantitation, measurements were done using the ZEN 2012 software and at least 94 cells were analysed per experimental condition.

Drosophila S2 cells were obtained from Life Technologies and were grown in Schneider's media containing 10% Fetal Bovine Serum. In order to process cells for microscopy, S2 cells were spotted onto concanavalin A (Sigma Aldrich) coated coverslips as previously described (Rogers and Rogers, 2008 (link)). For live imaging, cells were spotted onto glass bottom coverslip dishes (MatTek). In order to globally knockdown Sh3px1, S2 cells were treated with either control dsRNAs targeting gfp or with dsRNAs targeting sh3px1. The cells were processed for immunofluorescence or western blot analysis four days after dsRNA treatment. The dsRNAs were prepared and administered to the cells as described previously (Rogers and Rogers, 2008 (link)). DNA was transfected into S2 cells using Effectene (Qiagen) following the directions provided by the manufacturer. The shRNA constructs were also transfected using Effectene.

For gold nanoparticle imaging, 50% of the PEG on the GNP surface was replaced with a PEG-Cy5 complex (excitation 633 nm, emission filter 650 nm LP). The GNPs were prepared otherwise the same as described previously. Glass bottom coverslip dishes (MatTek) were used to facilitate live cell imaging. Cells were first incubated with the Cy5-labelled GNPs for 24 h with the dose of DTX elucidated before. Approximately 1 h before imaging, spheroids with incubated with NucBlue Live ReadyProbe Reagent (Invitrogen) which uses Hoechst 33342 to stain the nuclei. For monolayer, this was also completed 30 min prior to imaging. Image processing was performed using ImageJ (Schindelin et al. 2012 (link)).