Campbell and Moorehead BMC Cancer 2011, 11:480 http://www.biomedcentral.com/1471-2407/11/480 Page 8 of 13 Figure 4 Downregulation of PDGFRs in IGF-IR-independent recurrent cells increased cell number. MTT assays were performed to examine cell proliferation/survival after siRNA-mediated knockdown of PDGFRa and b alone or in combination (a). Cell number is expressed relative to transfection with the control oligonucleotide and represents the average of five trials. JC1 assays were used to assess apoptosis. Cells were plated in 12-well dishes and transfected with siRNA oligonucleotides specific to PDGFR. JC1 was added after 48 h and fluorescent green cells were quantified. Results represent the average of 3-4 trials relative to controls (b). Apoptosis was also evaluated by immunofluorescence staining for cleaved Caspase-3 (c). Proliferation was quantified using immunofluorescence staining for Ki67 (d) and phosphorylated HistoneH3 (e). For all three above assays, cells were plated in 6-well dishes on coverslips and transfected as above. Fixing and staining was performed 48 h post transfection. Images were collected at 100 × magnification and positive nuclei were counted. Results represent the average percent positivity relative to controls for three individual trials (d, e, f). * = p < 0.05 (relative to respective control). h, control cells had almost completely filled the wound; with knockdown of either PDGFRa or b, relatively large gaps were still observed and with dual knockdown, very little progress had been made in filling the wound (Fig- ure 5b). With knockdown of either PDGFR isoform, it took approximately 45 h for the wound to be completely filled, while with dual knockdown, wounds finally were filled by 72 h (data not shown). A system utilizing matrigel coated membranes was employed to assess the role of PDGFRs in extracellular