GLI1 in Ewing Tumors Figure 3. Multiple means of GLI inhibition, diminish ESFT cell line phenotype. Multiple GLI1 shRNA constructs (A, bottom) produce diminished GLI1 expression to varying degrees (Panel A, top) and diminished anchorage independent growth (Panel A, middle) in TC71. Panel B: GLI1 shRNA constructs produce the expected reduction in GLI1 protein by western blot in TC71. Panel C: Overexpression of the endogenous HH-GLI inhibitor Suppressor of Fused (SUFU) reduces anchorage independent growth of ESFT lines. doi:10.1371/journal.pone.0007608.g003 of the HH ligand or by a loss of function mutation in the Patched1 tumor suppressor. In EWS/FLI1 transformed NIH3T3, there is evidence for only minimal HH activation[4]. Furthermore, expres- sion data from primary Ewing tumor specimens does not demonstrate significant levels of HH ligand (Figure 1). To obtain further proof of the HH independence of GLI1 activation in ESFT, we assessed HH activation by the application of an active form of exogenous human SHH ligand to cells in culture. As shown on the left in Figure 4A, application of this ligand on the HH sensitive cell line NIH3T3 results in marked upregulation of GLI1 and of Patched1, demonstrating an intact HH-GLI transduction pathway with appropriate downstream signaling. The compound Cyclopa- mine, which blocks pathway activation at the level of Smoothened, effectively eliminates this activation as expected. In ESFT cell lines PLoS ONE | www.plosone.org 4 October 2009 | Volume 4 | Issue 10 | e7608