Genes were taken as hits if they had protocol a mean bootstrap in the upper quartile cutoff SI 0. 078 and the lower bound of 95% confidence interval 0. The results of a small sim ulation study we carried out show that the bootstrap dis tribution from a very small number of shRNAs is not reliable. Inhibitors,Modulators,Libraries Therefore, the mean SI value was calculated for the genes with 3 shRNAs. A more strin gent cutoff was used for hit selection among these genes. For the siRNA screen, the SI value was calcu lated by averaging the two siRNAs for each gene after normalization and the top hits for each cell line were selected based on the SI value of the averaged data. Cor relation between experiments was estimated using Pear sons correlation coefficient. Statistical analysis was performed using R software.
Cell growth and viability assays For cell growth assays cells were seeded at 5 �� 105 cells per well of a six well plate. The next day cells were treated with 5 uM CCT007093 or 10 nM mithramycin, 3 nM paclitaxel, or vehicle control. After three days cells were collected, washed, and counted using a Coulter Inhibitors,Modulators,Libraries Counter. Inhibitors,Modulators,Libraries Cell num ber was plotted Inhibitors,Modulators,Libraries as a percent of cells relative to vehicle control. Cell viability assays were performed by seeding 3,000 to 8,000 cells per well of a 96 well plate. The next day, growth media was replaced with treatment media containing vehicle DMSO or paclitaxel that was serial diluted by half log concentrations ranging from 0. 3 to 30 nM. After three days of incubation with the drug, cell via bility was measured using the Alamar Blue assay.
Cell viability for each drug concentration was compared to vehicle treated control. Four replicate wells from three independent experiments of each drug con centration were used to generate median effect plots to calculate the IC50 concentrations for each cell line using Calcusyn Software. Inhibitors,Modulators,Libraries IC50 values for each cell line are represented with standard error. Mammosphere cultures For three dimensional mammosphere cultures, cells were seeded on growth factor reduced Matrigel in chamber slides as previ ously described. CCT007093, mithramycin, and LY2109761 paclitaxel were added to medium 24 h after cell seeding and medium was replaced every three days. Mammospheres were detached from Matrigel with dis pase enzyme, trypsinized into single cell suspensions, and cell number was determined using a hemocytometer.
The number of viable cells was plotted as a percent of cells relative to vehicle control. Drug synergy analysis Regorafenib chemical structure Paclitaxel was combined with each of the different agents at a fixed ratio of the individual IC50 concentrations of each drug. Drug combinations were then serial diluted and represented as IC50, IC25, and IC12. 5 concentra tions, as the additive effects of both drugs. Statistical analysis of drug synergy was evaluated from the results of the Alamar Blue assays and calculated using the Chou Talaly method and Calcusyn Software.