Numb expression in human mammary tumors. (A) The typical immunoreactivity for Numb in normal breast (normal) showed intense staining in the vast majority of ductal (luminal) and lobular epithelial cells, with a prominent membranous staining pattern. Examples are shown of typical class-1(type-0), class-2, and class-3 tumors. Arrowheads point to normal glands within the context of the tumors. (B) Correlation between Numb status and clinical-pathological features. Details and explanations are in Table S1, available at http://www.jcb.org/cgi/content/full/jcb.200406140/DC1. Ki67, proliferative index; LN, lymph nodes. P value was obtained using the Mantel-Haenszel Chi square statistics. (C) In situ hybridization with an antisense probe for Numb mRNA was performed on paraffin sections. Control hybridizations with a corresponding sense probe gave no signal (not depicted). Examples of matching bright fields (top) and dark fields (bottom) of class-1 (left) and class-3 (right) tumors are shown. Numb transcripts appear as bright spots in the dark fields (bottom).

Re-expression of Numb selectively suppresses growth in Numb-negative tumors. (A) Primary tumor cells from class-1(type-0) (left) and class-3 (right) patients were transduced with retroviruses encoding GFP or Numb-GFP. After 3 wk, plates were fixed and stained (bottom). The histograms show the average number of colonies (colony-forming units ± SD) from three plates. Similar results were obtained with primary cultures from three independent class-1(type-0) and three independent class-3 patients (not depicted). (B) The expression of GFP and Numb-GFP, as detected by epifluorescence (top) or immunoblot (bottom), upon transient retroviral delivery is shown to demonstrate equal efficiency of infection/expression.

Effects of perturbation of Numb or Notch activity in mammary tumor primary cells. (A) Primary normal (left) or tumor (right) cells were transfected with siRNA oligos for Numb or control (ctr) oligos for 72 h and assayed for HES-1 mRNA levels (histograms) or immunoblotted with the indicated antibody (WB). (B) Primary tumor cells from class-1(type-0) and class-3 patients were transduced with GFP or Numb-GFP and assayed for HES-1 mRNA levels 72 h after infection (protein expression controls are as from Fig. 3 B, not depicted here). Normal primary cells from the same patients behaved as class-3 tumors (not depicted). In A and B, the mean fold induction (± SD) from two independent experiments performed in triplicate is shown. (C) Primary tumor cells from class-1(type-0) (left) and class-3 (right) patients were treated with DFP-AA for 10 d or mock treated (ctr), followed by staining (bottom) to count colonies. The histograms show the average colonies (colony-forming units ± SD) in triplicate plates. Results are representative of three independent experiments. (D) HES-1 mRNA expression from cells treated as in C, the mean fold induction (± SD) from two independent experiments performed in triplicate is shown. In all panels, results are representative of those obtained with primary cultures from three class-1(type-0) and three class-3 patients (not depicted).

Loss of Numb expression in tumors is due to enhanced ubiquitination and proteasomal degradation. (A) Matched normal (top) and tumor (bottom) primary cells from class-1 (right) and class-3 (left) patients were treated with MG132 (+) for 12 h or mock treated (−) and stained with anti-Numb. (B) Total cellular lysates from the same cells as in A were immunoblotted with anti-Numb (top). Molecular mass is indicated in kilodaltons on the right. Typically, two Numb-specific bands (each probably corresponding to a tightly spaced doublet) are detected in human mammary cells. Equal loading was checked with anti-actin (bottom). (C) Primary tumor mammary cells were either mock treated (−) or exposed to MG132 (+) for 12 h. Lysates were immunoblotted (WB) with the indicated antibodies. (D) Tumor mammary cells from class-1 and class-3 patients were either mock treated (−) or exposed to MG132 (+) for 6 h. Lysates were immunoprecipitated (IP) with a monoclonal anti-Numb antibody and immunoblotted (WB) with the indicated antibodies. Molecular mass is indicated in kilodaltons on the right. Results in all panels are representative of three independent experiments. In addition, similar results were obtained with primary cultures from three class-1(type-0) and three class-3 patients (not depicted).

Increased Notch signaling in Numb-negative tumors. (A) Primary tumor cells from class-1(type-0) (top) and class-3 (bottom) patients were treated with MG132 (+) or mock treated (−) for 1 h and stained with anti-Notch. Note the lower basal levels of Notch expression in class-1 MG132-untreated cells and the presence of nuclear Notch in the same class upon MG132 treatment. (B) CBF1-responsive reporter gene activity was evaluated in normal and tumor cells from class-1(type-0) and class-3 patients. (C) HES-1 mRNA expression in total RNAs from normal and tumor cells from class-1(type-0) and class-3 patients. In B and C, the mean fold induction (± SD) from two independent experiments performed in triplicate is shown. In all panels, results are representative of those obtained with primary cultures from three class-1(type-0) and three class-3 patients (not depicted).