Analysis of Shh signaling pathway components in breast cancer cells. A-D, expression of (A) Shh, (B) Ptch-1, (C) Smo, and (D) Gli-1 mRNAs in indicated breast cancer cell lines and a control cervical cancer (HeLa) cell line, was measured by qRT-PCR. Expression of individual genes is shown relative to the expression in HeLa cells, which is set to 1. Bars represent mean ± SD (n = 3). Experiments were performed twice in triplicate. E-I, indicated breast cancer cells stably expressing scrambled or Shh shRNAs were seeded at 5-7 × 104 cells/well, depending on cell type, in 6-well plates and cell numbers were quantified on day 6. J-N, indicated breast cancer cell lines stably expressing scrambled or Shh shRNAs were seeded at 1-2 × 104 cells/well in 24-well ultra-low adherence plates, and cell numbers were quantified 14 days later. For E-N, bars represent mean ± SD (n = 3). Three independent experiments were performed in duplicate using cells at three different passages. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; Student’s t test.