We used an ordinary differential equation model to charac terise the dynamic transitions between the four popula tions. We assumed that cells could enter and leave states with different, experiment dependent transi tion rates. Among the twelve theoretically possible tran sitions between different selleck chem states, we considered the six following ones, interphase cells may enter mitosis or die, mitotic cells may divide into twice as many interphase cells, become polynucleated or die, and polynucleated cells may die. We first considered a model with con stant rates, however, we found that the data from many of the movies could not be fit satisfactorily. There fore, we extended the model by allowing a simple time dependence of the transition rates, motivated by the notion that the effect of an siRNA on a cell population occurs with a time delay after the transfection, reflect ing differences in RNAi efficiency and protein life time.

Hence, to account both for experiment dependent pen etrance and delay of phenotypic effects, the transition rates were modelled with four parametric sigmoid func tions, each dependent on two parameters, a transition penetrance x and an inflection time point x. The same transition rate function kD was used for all three transitions into cell death. The interphase GSK-3 to mitosis kIM and mitosis to interphase kMI transi tion rates were modelled with non zero fixed intercepts, representing the basal rates in the untreated, prolifer ating populations. The model represents the temporal evolution of the four cell populations starting at cell seeding time, with an unknown initial number of cells n0.

To account for normal cell contamination, resulting from untransfected cells moving into the spot region, we introduced an additional contamination parameter u to represent the fraction of the cell subpopulation that fol lows a basal cell growth. Under this model, each spot experiment was described by 10 parameters, the initial number of cells n0 at seeding time, the contamination parameter u and 8 transition parameters, penetrance x and inflection time x each for kD, kIM, kMI and kMP. For each spot experiment, parameters were robustly estimated by fitting the cell count time course to the model by penalised least squares. The mean relative error, i. e. the merely average of absolute differ ences between the fitted and the measured cell counts relative to the maximum number of cells, measured the accuracy of the fit in one spot. 95% of the spot experi ments had an MRE lower than 3. 2%, demonstrating the overall high goodness of fit of the model. Spot experi ments with high MRE, indicative of lack of model fit, were discarded from the analysis.