Campbell and Moorehead BMC Cancer 2011, 11:480 http://www.biomedcentral.com/1471-2407/11/480 Page 6 of 13 Figure 2 PDGF receptors are inversely regulated with IGF-IR. Western blotting was used to examine expression in response to varying IGF-IR levels. Doxycycline was withdrawn from MTB-IGFIR mice with mammary tumors and tissue was collected 24 or 48 h later. Levels of PDGFRs were assessed and compared to that of tumors with IGF-IR induction (a). IGF-IR and PDGFR expression in a cell line (RJ345) created from a IGF-IR- dependent primary mammary tumor and two cell lines (RJ348 and RJ348) created from different IGF-IR-independent recurrent spindle tumors (b). Four cell clones expressing low levels of IGF-IR were generated through serial passage in the absence of doxycycline. Confirmation of IGF-IR downregulation as well as determination of PDGFRa and PDGFRb levels was performed (c). PDGFR levels in RM11A cells grown in vivo with (RM11A + dox) or without (RM11A-dox) doxycycline induction. Tissue was collected when tumors reached 17 mm in diameter (d). For all Western blots, b-actin served as a loading control. unchanged after PDGFR downregulation (Figure 3). Three trials were performed to confirm these findings. It was expected that given the dramatic increase in PDGFR expression in recurrent tumors and cells, this pathway would be important for cell proliferation and survival. To test this hypothesis, MTT assays were performed. Contrary to expectations, cell number increased by approximately 1.7 and 1.5-fold with PDGFRa knockdown in RJ348 and RJ423 cells, respec- tively. A more modest 1.4 and 1.2-fold increase in cell number was observed with downregulation of PDGFRb, while dual knockdown increased cell number by 2.0 and Table 1 Expression of PDGFR transcripts after suppression of IGF-IR levels RM11A cell line (in vitro) with doxycycline without doxycycline MTB-IGFIR transgenic (in vivo) Control 24 h doxycycline withdrawal 48 h doxycycline withdrawal *Statistically significant compared to control (with doxycycline) p < 0.05. IGF-IR 1.000 ± 0.000 0.042 ± 0.024 1.000 ± 0.223 0.010 ± 0.006 0.004 ± 0.002 PDGFRa 1.000 ± 0.000 1.608 ± 1.087 1.000 ± 0.392 3.113 ± 1.620 7.527 ± 2.475* PDGFRb 1.000 ± 0.000 2.953 ± 0.534 1.000 ± 0.210 3.193 ± 0.948 2.917 ± 0.818