Figure 4

Functional RNAi profiling of GPCR coding genes with prostate cell lines. (A) An antibody based assay for detection of nuclear Ki-67 and cleaved PARP was used for microscopic analysis of cell proliferation and induction of apoptosis (blue = DNA, red = cPARP, green = Ki-67). In comparison to DNA counterstaining the nuclear intensity for Ki-67 staining increases linearly from G1 to M cell cycle phase whereas the staining for cPARP increases similarly from induction of apoptosis to formation of apoptotic bodies. (B) For the analysis each array position was imaged using x20 objective. Cells were segmented on basis of DAPI counterstaining and the intensities of Ki-67 and cPARP were measured from the nuclear area. On the whole array level the markers displayed a mutually exclusive staining pattern (Bottom left) allowing robust delineation of proliferating and apoptotic cells (Bottom right). The green and red rectangles on the image display the gated populations of Ki-67 or cPARP positive cells respectively. (C) Hierarchical partitioning around medoids (PAM) clustering of the standardized (z-score) results of the two biological replicate CSMA experiments with LAPC-4, RWPE-1 and VCaP cells. (D) Top left: Scatter plot distribution of the replicate experiment z-score results for LAPC-4 cells. Top right and bottom: Line graph distribution of the combined replicate experiment results for each siRNA construct for the analyzed cell lines. SiRNAs inducing a greater than ± 2 z-score (displayed in red) were considered significant. Distribution of the negative control siRNAs shown in green.