Figure 4

Active Notch expression can bypass the Postn requirement for heterotopic growth of Met-1 cells. (A) Western blot analysis of tumor lysates showing a marked reduction of active Notch levels in Postn-null tumors. This is accompanied by a 2-fold reduction in the levels of Hey1 gene expression as measured by Q-PCR (B; ^* P <0.01). The data shown are an average of 4 independent tumors performed in triplicate. (C) Western blot analysis of Postn- or NICD-expressing Met-1 cells. Cells expressing Postn showed a marked increase in endogenous NICD levels when compared to vector control (pLPCX). The increase in endogenous NICD was blocked by the addition of a γ-secretase inhibitor (GSI). Overexpression of exogenous NICD was only detectable with a total Notch antibody as the anti-NICD epitope has been deleted in the construct. Similarly, expression of the NLS mutant can only be detected by Q-PCR as it lacks both epitopes recognized by the commercial anti-NICD and anti-Notch antibodies. (D) Quantitation of Hey1 mRNA levels showed a 4- and 70-fold increase in Met-1 cultures overexpressing Postn or NICD, respectively (^* P <0.01). No increase was observed in ΔNLS-expressing cells. (E) Tumor volume measurements at 28 days post-injection of Met-1 cells overexpressing NICD or ΔNLS in wildtype or Postn-null mice. Cells over-expressing NICD could bypass the Postn requirement for subcutaneous growth in FVB/N females. Little or no growth was observed for ΔNLS-expressing cells (^* P <0.01). (F) Expression of dnMAML1 in Met-1 cells impairs their growth in wildtype mice when compared to a GFP-expressing control (^* P <0.01).