Fig. 3.
Myc overexpression reduces Wnt expression creating paracrine dependency. (A) WB analysis for Wnt1 and actin protein levels in 2 representative WM+T and WMT control tumors. Bands were quantified by comparison with the loading control and are represented as fold change relative to average of the control tumors. (B) qRT-PCR for Axin 2 in normal mammary glands of ZP3-Cre;RCAG-MycERT2 mice or ZP3-Cre control mice (n = 5/5). (C) Quantitative real-time PCR for indicated targets in cellular extracts from 67NR-Myc-RFP cell lines treated for 24 h with doxycycline and either L-CM or L3-CM, both normalized to respective untreated controls with L-CM alone (n = 3). (D) Flow-cytometric analysis of cell death in 67NR-Myc-RFP cells after a 24-h treatment with doxycycline and either L-CM or L3-CM, both normalized to respective untreated controls with L-CM alone (n = 3). (E) Representative flow-cytometric analysis showing a dose-dependent retention of high-RFP/high-Myc–expressing 67NR-Myc-RFP clones during a 72-h treatment of doxycycline and mixtures of L-CM and L3-CM as indicated in the figure. (F) Quantification of β-catenin–positive nuclei on WM+T, WM−T control tumors, and mixed WM+T/WM−T tumors stained for β-catenin (n: Myclow = 4; Mychigh and mixed = 3). (G) Immunofluorescence for nuclear β-catenin in WM+T, WM−T control tumors and mixed WM+T/WM−T tumors. For box-and-whisker plots, the error bars represent min to max values, the box represents the interquartile range, and the horizontal line represents the median. P values are based on Student’s t test: *P < 0.05. For bar charts, error bars represent SD.