Figure 3.

Characterization of Numb phosphomutants. (A) Scheme showing the behavior of a SC (A) and daughter progenitor cell (B) in WT vs. Numb-KO (Tosoni et al., 2015). (B) Scheme of the growth of MS from WT and Numb-KO MECs (Tosoni et al., 2015). (C and D) WT and Numb-KO cells, transduced with the indicated constructs (DsRed fusion proteins; EV, empty vector), were assessed for SFE (C, by counting only red cells or MS) and size (D, N = number of epifluorescent MS analyzed). Results are expressed relative to WT cells (see also Table S2). Significance was calculated vs. EV cells. Representative images of the MS are in D, top panel. Bar, 100 µm. (E) WT and Numb-KO MS, transduced with the indicated constructs (Flag-tagged), were analyzed by IB. Arrows, endogenous (black) or overexpressed (red) Numb (also in G). Activated Notch (Act. Notch) was detected with the anti Val¹⁷⁴⁴ Ab (in this and all subsequent figures). Right: Quantitation of three independent experiments. (F) HEK-293 cells, transfected as indicated (all Numb constructs were Flag-tagged and also codify for an sh-RNA sequence against endogenous Numb; EV, empty vector), were IP and IB as shown. (G) HEK-293 cells were stably transduced with Notch-NΔE (Notch-TFX; NT, not transfected) and transfected with the indicated Numb-Flag constructs (as in F). IP and IB were as shown. (H and I) MCF-7 or Cal51 cells were either transduced with Notch-NΔE (Notch-TFX; I) or not (H). Cells were treated with BIS (or mock-treated) and IP and IB as shown. In H, IP-Ctr is anti-Flag; in I, IP-Ctr is goat IgG. Data are reported ± SD (C and E) or ± SE (D). Statistical analysis was with the Student’s t test two-tailed (C and D) or with the one-sample t test (E). Source data are available for this figure: SourceData F3.