Campbell and Moorehead BMC Cancer 2011, 11:480 http://www.biomedcentral.com/1471-2407/11/480 Page 5 of 13 Figure 1 IGF-IR independence is associated with upregulation of PDGFRs. Spindle recurrent tumors, formed after doxycycline withdrawal from MTB-IGFIR mice harbouring tumors and subsequent tumor regression, were compared to primary mammary tumors in which the IGF-IR transgene was expressed. Western blotting revealed an upregulation in PDGFRa and PDGFRb in spindle recurrent tumors (a). For the western blots, b-actin served as a loading control. Immunohistochemistry was also used to examine PDGFR expression in two different primary mammary tumors (b-e) and in two different IGF-IR independent recurrent tumors (f-i). Scale bar = 100 μm. IGF-IR when cultured in the absence of doxycycline both in vitro and in vivo [24]. In addition, the RM11A cells can be injected into the mammary fat pad of syn- geneic, FVB mice and these cells form mammary tumors faster in the presence of doxycycline than in the absence of doxycycline [23]. Evaluation of PDGFRa and PDGFRb in RM11A cells grown in vitro in the presence of doxycycline or in four independent clones grown in the absence of doxycycline for at least four passages revealed that both PDGFRa and PDGFRb levels increased in the clones expressing low levels of IGF-IR (Figure 2c). A similar increase in PDGFRa and PDGFRb levels was observed in RM11A cells grown in vivo in the absence of doxycycline compared to RM11A cells grown in vivo in the presence of doxycycline (Figure 2d). Downregulation of PDGFRs in IGF-IR-independent recurrent cell lines results in increased survival To further evaluate the function of PDGFRa and PDGFRb in mammary tumor cells that have become independent of IGF-IR signaling, PDGFRa or PDGFRb were knocked down alone, or in combination, in two IGF-IR-independent mammary tumor cell lines, RJ348 and RJ423. These two cell lines were chosen as they express high levels of PDFGRa and PDGFRb and low levels of IGF-IR (Figure 2b). Downregulation of PDGFRa or b alone or in combi- nation was consistently achieved in both cell lines and was validated up to 72 h post transfection (Figure 3). Knockdown of either receptor isoform did not result in downregulation of the other. Concomitant adminis- tration of siRNA for PDGFRa and PDGFRb was effec- tive at reducing the expression of both PDFGRs (Figure 3). Interestingly, with PDGFR knockdown, Akt activation increased in both recurrent cell lines (Figure 3). With pErk1/2, levels increased with PDGFR knockdown in RJ348 cells; however, in RJ423 cells, this protein was observed to have slightly decreased levels. Cyclin D1, an important molecule in our model [12,24], was