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Spheroids were monitored in real-time by live-cell imaging (Incucyte, Essen Instruments; 10x objective), acquiring 1 image/h.
Where indicated, nocodazole (20 µM) was added, and then cells were imaged by time-lapse microscopy, acquiring 1 frame/s for up to 10 min. Cells were tracked with Kinetic Imaging software (Andor Technology, Belfast, UK) and the trajectories were analyzed statistically using Mathematica software (Wolfram Research Inc, Champaign, IL).
Cells were stimulated either with 1% FBS alone or with other molecules for 15 min or 30 min, acquiring 1 image every 1 s or 3 s, respectively.
Cellular population analyses were performed with a FACS Calibur Becton Dickinson Cytometer (San Diego CA, USA) by acquiring 1 × 10 events (gated by forward and side scatter properties; in the case of intracellular staining, these parameters were adjusted accordingly) and analyzed using Summit Software (Beckmann Coulter; Brea, CA, USA).
After acquiring 1 kb promoter sequences of miRNA genes and their coexpressed genes in FASTA format, the Match program [ 35] was executed for sequence motif search of transcription factor binding sites according to the matrix information provided by TRANSFAC (version 2011.4).
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