Figure 3.
Ductal breast carcinoma in situ (DCIS) cell-dependent regulation of platelet derived growth factor receptor (PDGFR) expression in fibroblasts. Representative immunoblots of PDGFR expression in human mammary fibroblasts (HMF) and results from corresponding quantifications of (A–C) PDGFR protein and (C and D) PDGFR mRNA under conditions of (A) HMF exposure to control medium or DCIS cell medium (n = 4), (B) trans-well HMF cultures with or without DCIS cells (n = 3), (C) mono-culture or co-cultures of green fluorescent protein (gfp)-labeled HMF with MCF10A normal breast epithelial cells or breast MCF10A.DCIS cells (n = 4), and (D) HMF-gfp cultured either on fixed HMF or fixed DCIS cells (n = 7). Protein levels (immunoblot data) were determined using β-actin (A and B) or gfp (C) as loading control and further normalized to the mean of the control group (set to 1.0 arbitrary units [a.u.]). Data are presented as average with standard deviation (SD). For PDGFRα and -β detection, the membranes were stripped and reprobed. β-Actin or gfp was detected on the same membrane. Changes in gene expression (quantitative real-time PCR data; C and D) were calculated by the comparative ΔΔCT-method with GFP as housekeeping gene for input control. Expression values were further normalized to the mean of the control monoculture HMF (set to 1 a.u.) and are represented as average with SD. P values were derived from ANOVA with Bonferroni post hoc test. (E) Microphotographs of immunohistochemistry-detected PDGFR expression in orthotopic tumors formed after injection of DCIS cells into the mammary fat pad of SCID mice. Images were adjusted for presentation. Size bars are 100 μm.