FIG 1.
DTX1 negatively regulates NOTCH1/HES1 signaling and suppresses invasion of osteosarcoma cells. Immunoblot (A) and PCR (B) analysis of DTX1, NOTCH1/ICN1, HES1 and actin in OS187 cells stably expressing vector control or DTX1 gene. To confirm that the band identified by the western blot for ICN1 in fact represents the cleaved intracellular fragment of Notch1, we cultured OS 187 cells in 10 or 50 μM compound E, a gamma secretase inhibitor (GSI), and showed that the cleaved protein is reduced with GSI treatment (Supplemental figure 1). (C) Histograms represent quantitative PCR analysis of NOTCH1, HES1 and DTX1 from the same cells. Ct values were normalized to actin; vector control is defined as a relative expression of 1 (*, p<0.05). (D) Histograms represent luciferase emission from a CSL reporter (i.e., NOTCH-responsive) in OS 187 cells transduced with DTX1 or empty vector (*, p<0.05). (E) Histograms represent matrigel invasion of OS187 cells stably expressing empty vector, DTX1 or DTX1 and HES1. 1×104 stably transduced OS187 cells were seeded in each transwell and cell invasiveness was quantified by the invasive cell number after 48 hours incubation. Average of three wells, with error bars showing standard deviation, is shown (*, p<0.05).