NOTCH1 inhibition results in mammary tumor regression in vivo and reduces tumorsphere activity in vitro. (A) NOTCH1 inhibition in vivo results in rapid mammary tumor regression. Tumor-bearing mice (n = 5) were treated with doxycycline (10 μg/ml) for 11 days, and tumor volume was monitored externally by using calipers. The graph shows average tumor volume over time and standard error. (B) Doxycycline treatment results in decreased NOTCH1 target-gene expression. Total RNA was harvested from mammary tumors isolated from mice left untreated or treated with doxycycline (10 μg/ml) for 24 or 48 hours. Hes1, Deltex1, and c-Myc expression levels were quantified by using real-time quantitative PCR. Results were plotted relative to untreated levels. (C) NOTCH1 inhibition in vivo interferes with disease recurrence. Tumor-bearing MMTV-tTA/TOP-ICN1 transgenic mice were administered water containing doxycycline (10 μg/ml) for 28 days, and tumor volume was monitored externally by using calipers. Doxycycline was then removed from the water, and mice were monitored for tumor regrowth for an additional 40 days. Mice that failed to exhibit tumor regrowth were then killed and examined histologically for the presence of residual mammary tumor cells.