Snail

NVP-LDE225 and NVP-BEZ-235 were obtained from Chemie Tek (Indianapolis, IN). Antibodies used against Gli1, Gli2, phospho-Akt, Akt, caspase-3, PARP, E-Cadherin, N-Cadherin, Vimentin, Sox-2, Oct-4, Nanog, c-Myc, phospho-Akt, phospho-PI3K, phospho-p70S6K, phospho-GSK3Kβ, phospho-4EBP1, Akt, p70S6K, GSK3Kβ, 4EBP1, Slug, snail, snail, patched 1, patched2, Smo, Sufu Bcl-XL, Bax, Bak, Bcl2, and β-actin were purchased from Cell Signaling Technology, Inc. (Danvers, MA). Enhanced chemiluminescence (ECL) Western blot detection reagents were purchased from Thermo Fisher Scientific Corporation (Waltham, MA).
Isolation and characterization of CD44⁺CD24⁺ESA⁺ pancreatic CSCs have been described elsewhere [54 (link)]. Pancreatic CSCs were grown in well-defined stem cell culture medium [54 (link)–56 (link)].

Total protein was extracted from lung or A549 cells and homogenized in RIPA lysis buffer with protease inhibitor and phosphatase inhibitor cocktails before being quantified using a BCA protein assay kit (Shanghai EpiZyme Biotechnology Co., Ltd.). Total protein extraction was resolved by 10% acrylamide gel before being transferred to the nitrocellulose membrane. The nitrocellulose membrane was blocked for 1 h at room temperature with 5% non-fat milk. They were then incubated overnight at 4°C with the following specific primary antibodies: N-Cadherin (1:1,000), E-Cadherin (1:1,000), Vimentin (1:1,000), p-Smad2/3 (1:1,000), Snail (1:1,000) (all from Cell Signaling Technologies, Danvers, MA), α-Smooth muscle actin (α-SMA) (1:1,000, SinoBiological Inc., Beijing, China), Collagen I (1:1,000, Bioss, Beijing, China), and β-actin (1:5,000, Sigma-Aldrich). Finally, the membranes were incubated for 1 h at room temperature with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies before exposing them to enhanced chemiluminescent reagents (Solarbio^® Life Science, Beijing, China).