Campbell and Moorehead BMC Cancer 2011, 11:480 http://www.biomedcentral.com/1471-2407/11/480 Page 7 of 13 Figure 3 siRNA mediated downregulation of PDGFRs. RJ348 and RJ423 recurrent cell lines were plated and 48 h later they were transiently transfected with oligonucleotides specific for either PDGFRa, b or both; an appropriate GC oligonucleotide was used as a control. Seventy-two hours post-transfection, protein was collected and subjected to western blotting probing for both PDGF receptors as well as pAkt, Akt, pErk1/2, Erk1/2 and cyclin D1. b-actin served as a loading control. 1.8-fold in RJ348 and RJ423 cells, respectively (Figure 4a). To determine if this effect was due to changes in proliferation or apoptosis, subsequent assays were performed. Apoptosis was addressed by using both JC-1 assays and staining for cleaved caspase-3 after knocking down PDGFRs. JC1 assays are based on the detection of loss of mitochondrial membrane potential, a hallmark of apoptosis, using a lipophilic cationic dye 5,5’, 6,6’-tetra- chloro-1,1’,3,3’ -tetraethylbenzimidazolcarbocyanide dye (JC-1); this dye naturally exhibits a green fluorescence during low membrane potential, but changes to red fluorescence as membrane potential increases. Thus cells staining intensely green are counted as apoptotic [26]. As seen in Figure 4b and 4c, knockdown of PDGFRa resulted in a substantial decrease in apoptosis in both cell lines, while PDGFRb downregulation resulted in a more modest insignificant decrease in apoptosis. Little further decrease in apoptosis, compared to PDGFRa knockdown, was observed when both iso- forms were targeted as assessed through both aforemen- tioned assays (Figure 4b, c). Proliferation after PDGFR downregulation was quanti- fied through staining for Ki67, a well-known marker of proliferation, and phosphorylated HistoneH3. As seen in Figure 4d and 4e, PDGFRa as well as dual PDGFR knockdown resulted in an almost identical increase in relative Ki67 and pHistoneH3 positivity of approximately 1.6-fold and 1.3-fold respectively in both cell lines. In RJ348 cells, PDGFRb had no effect on Ki67 positivity. In RJ423 cells, knockdown of this receptor isoform had vari- able results with an average overall increase in Ki67 staining of approximately 1.8-fold (Figure 4d). PDGFRb knockdown had only a minimal effect on phospho-Histo- neH3 staining in both cell lines (Figure 4e). Only down- regulation of both PDGFR isoforms resulted in a significant increase in proliferation (Figure 4d, e). PDGFR knockdown in recurrent cell lines impairs migration and invasion To assess the importance of PDGFRs for cell migration in these recurrent cell lines, scratch wound assays were employed after downregulation of PDGFRs. Images were captured at various timepoints after the scratch wound was made and images shown are representative of four trials (Figure 5). As shown, in the RJ348 cell line, con- trol cells had penetrated the wound by 14 h and wounds were observed to be virtually filled by 28 h. With knock- down of either PDGFRa or b, a slight impairment was observed after 14 h; however, after 28 h no differences were seen compared to control cells. When both recep- tor isoforms were downregulated, cells did not begin to fill the wound until after 14 h and even after 28 h, many large gaps devoid of cells were observed (Figure 5a). For RJ423 cells a similar trend was observed; a dra- matic reduction in migration was observed after knock- ing down individual PDGFR isoforms or both isoforms at the first timepoint, 16 h. At the second timepoint, 40