Genes that modulate two-dimensional co-culture growth are also important in anchorage-independent three-dimensional growth. Three-dimensional Matrigel culture assays were performed with 184-hTERT-L9 cells after they were transfected with small interfering RNA (siRNA) pools targeting the 47 genes required for mammary epithelial cell growth. The acini (with an acinus defined as a ball of 50 cells or more) formed per well were counted 8 days postplating, and surviving fraction estimates were generated in comparison to both transfection reagent-only and nonsilencing siRNA controls. Acini count fold changes are plotted as a rank relative to the control mean. Fold changes were calculated as mean number of acini when the gene was silenced in three-dimensional cultures divided by the mean number of acini when siRNA and transfection reagent controls were used in three-dimensional cultures. Error bars represent 95% confidence intervals (n = 4).