Campbell and Moorehead BMC Cancer 2011, 11:480 http://www.biomedcentral.com/1471-2407/11/480 anti-IGF-IR (R&D Systems, Minneapolis, MN, USA) all used at 1:1000 as well as anti-b-actin (Cell Signaling Technology, Danvers, MA, USA) used at 1:5000. Sec- ondary antibody was anti-rabbit IgG (Cell Signaling Technology, Danvers, MA, USA) or anti-Goat (Santa Cruz, Biotechnologies, Santa Cruz, CA) used at 1:2000. Quantification of protein levels was performed using a FluorChem 9900 imager and AlphaEaseFC software ver- sion 3.1.2 (Alpha Innotech, San Leandro, CA, USA). Immunohistochemistry Immunohistochemistry was performed as described in [25]. Sections cut from primary and IGF-IR-devoid recurrent tumors described in [13] were used. Antibo- dies used were anti-PDGFRa and anti-PDGFRb; both were used at 1:100 (Cell Signaling Technology, Danvers, MA, USA). Biotinylated anti-rabbit secondary antibody (Sigma-Aldrich, St. Louis, MO) was used for both at a concentration of 1:200. Images were captured using light microscopy. Real time PCR Real time PCR was performed to quantify transcript levels. RNA was isolated using a mirVANA kit (Ambion, Streetsville, ON) in accordance with the manufacturer’s instructions (without the miRNA enrichment step). Reverse transcription and real time PCR was performed as described in [13]. Primers specific genes encoding human IGF-IR [13], as well as PDGFRa and PDGFRb (both obtained from Origene, Rockville, MD) were used. The hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene was used for normalization. Transfections and siRNA-mediated protein knockdown Transient transfections were performed using Lipofecta- mine (Invitrogen, Burlington, ON) in accordance with the manufacturer’s directions. RNA oligonucleotides were transfected at a final concentration of 200 pmol/ mL. Briefly, three Stealth siRNA oligonucleotides with binding specificity for murine IGF-IR, PDGFRa or PDGFRb mRNA (Invitrogen, Burlington, ON) were ori- ginally tested for their ability to diminish expression of these gene products through western blotting. An appropriate GC control oligonucleotide (Invitrogen, Bur- lington, ON) was used as a negative control. MTT survival assays MTT survival assays were performed as described in [24]. Briefly, 500 cells/well were plated in 96-well plates in tri- plicate. Two days after seeding, cells were transfected with siRNA as indicated above. Three days later, MTT was added to the cells which were subsequently lysed after 1 h. Values are reported relative to the correspond- ing GC control and represent the average of 5 trials. Page 3 of 13 Immunofluorescence Immunofluorescence was performed as described in [25]. To assess proliferation, cells plated on coverslips were transfected with siRNA oligonucleotides specific for PDGFRa, PDGFRb or both as described above. Forty-eight hours after siRNA treatement, cells were fixed with 10% formalin for 1 h at room temperature and stained for Ki67. Primary antibody, anti-Ki67 (Abcam, Cambridge, MA), was used at a concentration of 1:200, while antibodies specific for cleaved Caspase-3 and phospho-HistoneH3 (both from Abcam, Cambridge, MA) were both used at 1:100; fluorescent conjugated Alexafluor secondary antibody (Invitrogen, Burlington, ON) was used at a dilution of 1:2000. Images were taken using using an Olympus BX61 fluorescent micro- scope (Center Valley, PA) with MetaMorph version 7.6.0.0 software (Molecular Devices, Downington, PA) at a magnification of 100×. Positive nuclei from 5-10 fields of view were counted; results represent the average posi- tivity compared to respective controls from three inde- pendent trials. JC-1 assays JC-1 assays were employed to test apoptosis after downregulation of PDGFRs. Recurrent cell lines were plated in 12-well plates. Twenty-four hours later cells were transfected with RNAi as outlined above. Forty- eight hours post-transfection, media was removed and fresh media containing JC-1 (Invitrogen, Burlington, ON) at a concentration of 200 μg/mL was added to the cells. Cells were incubated with JC1 for 15 min at 37°C, after which media was removed and wells were washed three times with PBS. Images were captured using an Olympus IX81 microscope (Olympus, Mark- ham, ON) at 200 × magnification using Image Pro Plus V.5.0.2.9 software (MediaCybernetics, Bethesda, MD) with the green fluorescent protein (GFP) channel (485-535 nm). Scratch assays For scratch tests, cells were plated in 12-well plates such that they were 90-100% confluent at the time the scratch was performed. Transient transfection with siRNA (as stated above) was performed 48 h after the cells were seeded. A single scratch was made down the center of the well with a 200 μL pipette tip 4 h post transfection. Subsequently media was removed, cells were washed twice with PBS and media was replaced. Cells were imaged at various timepoints (including immediately after the scratch was produced, 0 h) using an Olympus IX81 microscope (Olympus, Markham, ON) at 100 × magnification and Image Pro Plus V.5.0.2.9 software (MediaCybernetics, Bethesda, MD). This experiment was replicated four times.