EMT and claudin-low cells are insensitive to Hedgehog (Hh) pathway inhibitors. (A) Inhibition concentration (IC)50 values obtained with other Hh pathway antagonists from the 384-well screen. NF indicates not determined, either because no curve fit could be obtained, or the IC50 was outside the experimental concentrations tested. (B) GLI1 transcript levels in HMLE-shEcad and HMLE-shGFP cells treated with cyclopamine (0, 5, or 10 μM for 72 hours). (C) Immunoblot of GLI1 in HMLE cells treated with cyclopamine as in B). (D) Gli1 transcript levels after 16 h treatment with 1 μM JK184 or triptolide. Error bars indicate the standard error of two independent experiments. * = P <0.05, t test. ** = P <0.005, t test. (E) Anti-GLI1 immunoblot following treatment described in A). Lanes 1 to 3 are from a separate gel. Quantification is normalized to no treatment condition. EMT, epithelial-to-mesenchymal transition; GLI1, glioma-associated oncogene 1; HMLE, human mammary epithelial cells, transformed with large T and small t antigens.