Fig. 5. Phenotypic analysis of EML3 knockdown using time-lapse imaging. (A) siRNA oligos specific for Eml3 (80 nM) were used to knockdown the respective mRNAs in HeLa Kyoto cells; the same concentration of scrambled, nontargeting siRNAs served as a control. Left panel, qRT-PCR quantification of siRNA-mediated gene knockdown 24 hours post transfection (see experimental procedures). The relative remaining levels of target mRNA expression normalised by GAPDH mRNA was measured compared with cells transfected with scrambled siRNA oligos. Error bars represent the maximum and minimum expression levels of three individual experiments. Right panel, total cell lysate was prepared from HeLa cells before and after knockdown of EML3 using two different siRNA oligonucleotides (1 and 2) and EML3 was detected by immunoblot using specific antibodies. Tubulin served as a loading control. (B) siRNA oligos were used to knockdown EML3 in HeLa Kyoto cells stably expressing Histone 2B-EGFP. 29 hours after transfection, the EGFP signal was recorded at intervals of 30 minutes for another 48 hours. Automatically analysed phenotypes were classified and indices of mitosis, apoptosis, shape and the overall proliferation rate plotted over time. Results of representative experiments are plotted in blue (continuous line, fitted curve; dotted line, measured data points) and the control confidence band (mean ± s.d.) in grey. (C) The EGFP signal was recorded for 14 hours at intervals of 5 minutes, 60 hours after siRNA oligo transfection. Sections from selected frames were used for still images, times after seeding (i.e. transfection of) the cells (in brackets) and time intervals from the respective starting points are indicated.