For the DIV and nNRM indices in this assay, to achieve a power level of 0.8 requires 900 and 1100 cells respectively. CV?=?30% for all those unimodal distributions. The 2 2 bimodal distributions combine the distributions with means of 500 and Rhod-2 AM 10 and CV?=?30%, equally weighted (500+10) and with a 9010 split (500(90%)+10(10%)). Each distribution consists of 2000 random numbers. B) The same distributions as in A, logarithmically scaled. The key defines the reference points labeled around the plot (A).(TIF) pone.0102678.s002.tif (978K) GUID:?9376E25C-BA10-4D6A-9341-63029FDB6FFC Physique S3: Quantitating the Reproducibility of Intensity Measurements by Flow and Imaging Cytometry. A) Histogram of the total intensity of 2 m flow cytometry standard beads measured by flow cytometry shows peaks for single beads (red, CV?=?2.8) and double beads (blue). B) Histogram of the same beads centrifuged in the wells of a 384 well microplate and imaged to measure total bead intensity also shows peaks for single beads (red, CV?=?5.2) and double beads (blue). C) The histogram of total nuclear intensity in Cal33 cells fixed in suspension, labeled with Hoechst and measured by flow cytometry. Cell cycle modeling identifies 3 subpopulations, G0/G1 (red), S (hashed), and G2/M (blue). D) The histogram and cell cycle modeling of the same cells centrifuged in Rabbit Polyclonal to Shc (phospho-Tyr349) the wells of a 384 well microplate then imaged and analyzed for total nuclear intensity.(TIF) pone.0102678.s003.tif (238K) GUID:?9A8DEB99-AEE7-4C26-9959-10DBF47D1FBB Physique S4: The distributions of STAT3 activation for cell cycle subpopulations. Histograms of STAT3 activation in unstimulated and IL-6 stimulated Cal33 cells (the cell cycle states identified in the legend). Inset DNA histogram of the cumulative populace shows the mapping of DNA intensities to cell cycle says.(TIF) pone.0102678.s004.tif (544K) GUID:?878C9702-DD9B-4EB9-B31D-28DD9F8472CE Physique S5: Western Blot Analysis of Receptor Expression. A) Western blots of receptor expression in five cell lines, with Tubulin as Rhod-2 AM a control. B) Quantitation of total density in western blot bands for the IL-6 receptor, relative to Cal33 cells. C) Quantitation of OSM receptor expression, relative to Cal33. D) Quantitation of IFN receptor expression, relative to Cal33.(TIF) pone.0102678.s005.tif (517K) GUID:?6C868C84-FB1A-442D-9121-13621F0F6A6C Physique S6: STAT3 activation by Combinations of Cytokines. To assess the conversation between the IL-6 and OSM pathways, Cal33 cells were exposed to combinations of IL-6 and OSM for 15 min. Top Row) The activation of STAT3 by IL-6 alone. Left Column) The activation of STAT3 by OSM alone. The arrows point to the location of the population non-responsive to IL-6. Note that with the addition of OSM all cells treated with IL-6 show activated STAT3.(TIF) pone.0102678.s006.tif (599K) GUID:?71CE9286-9EAA-4DF0-818C-EB84EF188203 Figure S7: Evaluation of Potential indices of Diversity and Normality. A) Model distributions and cell data were used to evaluate the performance of selected metrics for characterizing the distributions. The 5050 mix consists of 2 unit normal distributions of equal populace that are separated by d standard deviations (sd). The 9010 mix consists of 2 unit normal distributions with 90% and 10% of the population, separated by d standard deviations. B) Selected Diversity and Normality indices were used to evaluate the model distributions for values of d ranging from 0C10 sd, and the Cal33 data for IL-6 and OSM stimulation. For the Cal33 data, the diversity indices all show similar response, while the model data show some key differences. The IQR (interquartile range) is not sensitive to the small 10% subpopulation, and the SE (Shannon entropy) and DE (Differential entropy, the Shannon entropy for a continuous distribution function) both plateau when the 2 2 populations individual. Only the QE (Quadratic entropy) shows a steady increase for both distributions. Again, the general pattern of the Normality steps Rhod-2 AM is similar for the Cal33 data, but the model data show key differences. The skewness and MMR (mean/median) are insensitive to the 5050 populace because it is usually symmetric, The kurtosis and KS statistic are sensitive to the variation in both distributions, however the KS was favored due to its direct interpretation as a measure of normality.(TIF) pone.0102678.s007.tif (1.0M) GUID:?BEB2DD7A-E027-4DFE-9B6B-C6FA05A35E75 Figure S8: Dose dependence of inhibitors of STAT3 activation by IL-6. STAT3 activation in Cal33 cells is usually inhibited by Pyridone-6 (?) with an IC50?=?66 nM or STATTIC (?) with an IC50 ?=?10 M.(TIF) pone.0102678.s008.tif (602K) GUID:?70EE9220-D3D6-4368-BDED-911F845DCDA8 Figure S9: Integrating heterogeneity analysis into phenotypic screening. Heterogeneity indices are evaluated during assay development and thresholds decided based on the goals of the project. For drug discovery and cell/tissue profiling programs that encounter phenotypic heterogeneity, HCS images are analyzed to generate the features, statistical parameters and HI’s. For samples or treatments with low Diversity (DIV) or a normal distribution (low nNRM) standard statistics can be used. A well or sample with a high HI and high nNRM or high.