We carried out RT-PCR to determine which concentration of GSI X effectively inhibited Notch activity in osteosarcoma cells, and then measured the expression of the Notch pathway target HES1. In 143B cell, GSI X at 5 μM reduced mRNA levels of HES1 in 143B cells more than 60% (Figure 2A). The gene discussed is HES1; the disease is osteosarcoma.