Fig. 6. Notch delays anoikis and promotes proliferation by augmenting NF-κB activity.

(a) Inhibition of luminal prostate sphere formation by BAY-11-7085. Inset image: Western blot analysis of pho-P65. (b) Super IκBα suppresses luminal sphere formation. Data represent means ± s.d. from 3 independent experiments. (c–d) Quantitation of sphere-forming units (c) and prostate sphere types (d) from prostate cells of K8-IKKβ mice treated with tamoxifen (Tmx) and vehicle (Veh). Data represent means ± s.d. from 3 independent experiments. (e) Comparison of sizes of PB-NICD and K8-IKKβ luminal prostate spheres. Dot graph shows means ± s.d. from one of the two independent experiments. Scale bars, 25μm. (f) Western blot of expression of Bcl-2, Bcl-xL and cIAP1 in FACS-sorted prostate luminal cells from WT, PB-NICD and PB-IKKβ mice. (g) Western blot of Pten expression and AKT activity in FACS-sorted prostate luminal cells from WT, PB-IKKβ and PB-NICD mice. (h) qRT-PCR and Western blot of Cyld expression in NICD^WT and PB-NICD mouse prostates. qRT-PCR data represent means ± s.d. from 3 independent experiments. (i) Expression of dominant negative Hes1 (dN-Hes1) in PB-NICD cells upregulates Cyld expression but does not significantly affect luminal sphere formation and size. Dot graphs show data representing means ± s.d. from 3 independent experiments. (j) Western blot analysis of Pten expression and AKT activity in luminal spheres derived from PB-NICD cells infected by GFP and dN-Hes1 lentivirus. (k) Ectopic expression of Hes1 in FACS-sorted PB-IKKβ prostate luminal cells enhances AKT activity and increases luminal sphere forming unit and sphere size. Dot graphs show data representing means ± s.d. from 3 independent experiments. (l) Schematic illustration of molecular mechanism through which Notch and NF-κB promote formation of luminal spheres. Boldness of arrow line reflects signaling contribution. Statistical significance was calculated using the t-test (**: p<0.01, ***: p < 0.001).