and A549 cells were cultured in DMEM (Cell-Gro, 10-013-CV) supplemented with 10% FBS (Atlanta Biological, #S11150). To obtain stable cell lines expressing H2B-GFP, A549 cells were infected with retroviral particles containing the H2B-GFP sequence under transcriptional control of the immediate early CMV promoter. Two days after infection, the cells were selected for expression with G418 at the lowest concentration that caused death of uninfected control cells within one week. Cells were cultured at 37uC in 5% CO2 in a humidified environment. Cell Treatment Cells were plated in sterile 24-well optical bottom plates (Greiner bio-one #662892) in 500 mL culture medium (35,000 cells per well for siRNA transfection; 45,000 cells per well for chemical treatments). After 24 hours of plating, the cells were either transfected with 200 nM of the siRNAs listed below using OligoFectamine (Invitrogen), following the manufacturer’s proto- col or treated with drugs at indicated concentrations. Cells were imaged 24 hours after siRNA transfection or immediately after chemical treatments at indicated time intervals. MAD2 siRNA sense strand: 59- GGA ACA ACU GAA AGA UUG G UU-39 (Dharmacon, custom sequence). BubR1 siRNA sense strand: 59- GGA AGA AGA UCU AGA UGU A UU-39 (Dharmacon, D- 004101-01). Non targeting control siRNA#3 sense strand: 59 AUG UAU UGG CCU GUA UUA G UU-39 (Dharmacon, D- 001210-03). Image Acquisition Images were acquired with Nikon Elements (v2.3 and above) on an automated epifluorescence Ti Eclipse microscope with Perfect Focus System (Nikon Instruments Inc, USA) with a motorized Prior XYZ-plane stage, a Hamamatsu ORCA-ER digital camera and a 206 objective (PLAN APO, 0.75). An X-Cite light-source was used with 2 neutral density filters (ND4 and ND8) allowing 1/4th and 1/8th (1/32nd overall) of light to go through. In addition, one field diaphragm and one aperture diaphragm present in the path of excitation light were set so as to keep the area exposed to light and the light intensity, respectively, to a minimum. The light filter set was carefully selected to strongly limit exposure to UV light (chroma filter set 49002: dichroic T495LP, excitation filter ET 470/40X, emission filter ET 525/50m). A custom designed microscope incubator set at 37uC with injection of humidified air/CO2 5% gas mix was used to maintain environmental control. Cells were imaged using 200 msec exposure time and 262 binning resulting in an image size of 6726512 pixels. HeLa cells were imaged every 4– 12 minutes, giving a total of 241–721 images per position while A549 cells were imaged every 5 minutes. Each movie was then exported in a separate folder from NIS Elements as uncompressed (12 bit) TIFF files to an 18 Terabyte network attached storage array for processing. Manual measurement of mitotic and interphase duration Manual measurement of mitotic duration was performed by one experienced individual to ensure consistency. Images were analyzed in ImageJ (NIH). Mitotic duration was defined as the difference between the mitotic entry frame and the first frame showing chromatid segregation for each mitotic event visible throughout a movie. The frame of mitotic entry was defined as the first frame with nucleus showing early prophase characteristics, such as initiation of chromatin condensation. If an advanced prometaphase cell was identified as the first mitotic event, then the prior (interphase) frame was chosen as the mitotic entry to avoid underestimating mitotic duration. Each entry and exit frame as well as the mitotic duration were recorded in excel files and the Automated Cell Cycle Analysis data was used to calculate the median and mean event duration and to plot the cumulative frequency curves, using the Tools/Data analysis/Histogram function of Excel, for each condition tested in an experiment. Interphase duration was determined to be the time difference between the first frame of segregation of the first mitosis to the mitotic entry frame of the following mitosis. Typically half of the movies analyzed automatically were analyzed manually, to limit time of analysis to a reasonable amount while ensuring good population representation. Processing and Data Output DCELLIQ was run on all movies with default parameters, unless otherwise stated, with time-series or SVM phase identification as indicated, using MATLAB (The Mathworks, versions 7.5.0, 2007b to 2010b) on a Dell Precision 380 with Intel Dual P4 3.0 GHz with 3 GB of RAM. Each image series analysis data is output into files containing: duration of mitotic events, mitotic entry time in a movie for each mitotic event, cumulative mitotic duration data, duration of interphase events, interphase start time for each interphase event measured, and cumulative interphase duration data. In addition, cumulative frequency curves for mitotic duration are generated by the DCELLIQ graphical user interface. DCELLIQ and a user manual are freely available for download here: http:// www.cbi-tmhs.org/Dcelliq/. Statistical analysis For comparison of differences between independent sets of measurements, the Mann-Whitney-Wilcoxon non-parametric test was performed. Significance level was alpha = 0.05. The Mann- Whitney-Wilcoxon test relies on comparing the ranks of sample measurements of two conditions rather than comparing the sample measurement values. The measurements of both samples are ranked together. Under the hypothesis, the distribution of the two measurement samples is the same. Under the alternative hypothesis, there would be a difference in distribution of the two measurement samples, so measurements from one sample population would precede measurements from the second sample in the distribution. The distributions in the two sample groups were considered significantly different when p,0.05. Statistical analysis was performed using the statistics analysis package program jmp 8.0 (SAS Institute Inc.). In some instances where the p value was lower than an arbitrary cutoff of 2610216, we report the values as #2610216. Segmentation, Tracking and Feature Extraction Nuclei were segmented from the background as described previously [11]. The centroid for each nucleus was calculated in each frame. Starting from frame one, possible matches for each nucleus were identified within a 30 pixel radius. Best matches were identified based upon similarity of area, grey value histogram, XY displacement, speed, direction, shape and relationship with neighbors (Delaunay triangulation). Wavelet, geometric, moment, texture and shape descriptor features can be extracted from each nucleus identified for a total of 211 features for the SVM approach. Wang et al. fully reference the 211 features [11]. The time series involved analysis of only 11 geometry features (included in the 211 features used in the SVM approach) and Area and Intensity were the only features necessary for identifying the transitions between interphase and mitosis. Description of Time-Series Algorithm The approach begins with the evaluation of the area, A, as a function of time (Figure S4a). Because mitotic division is associated PLoS ONE | www.plosone.org 11 September 2011 | Volume 6 | Issue 9 | e25511