Fscn1

After transfection, the cells were washed with ice-cold PBS three times and lysed with radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) supplemented with protease inhibitors (Promega Corp.). Thirty micrograms of proteins was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to the nitrocellulose membrane. The membrane was blocked with 5% nonfat milk and incubated with the primary antibody for 1.5 h. The protein band, specifically bound to the primary antibody, was detected using the FluorChem imaging system (Alpha-Innotec GmbH, Kasendorf, Germany). The primary antibodies were FSCN1, ERK, and p-ERK (both 1 : 1,000 dilution; both Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and GAPDH (1 : 5000; #5174, Cell Signaling Technology).

Total cellular protein and western blot analysis were performed according to previous studies [43 (link), 44 (link)]. The antibodies used were as follows: Dicer (#5362; Cell Signaling), E-cadherin (#610181; BD), FSCN1 (sc-46675; Santa Cruz), GAPDH (sc-365062; Santa Cruz), LASP1 (MAB8991; Merck Millipore), Vimentin (#550513; BD) and XIAP (#14334; Cell Signaling).

Formalin-fixed, paraffin-embedded tissue was cut into 4 μm section, de-paraffinized in xylene, rehydrated through graded ethanol, quenched for endogenous peroxidase activity in 3% hydrogen peroxide, and processed for antigen retrieval by heating in 10 mM citrate buffer (pH 6.0) at 90–100°C (XIAP, FSCN1, Ki-67, Bcl-2, Bax, Caspase-3). Sections were incubated at 4°C overnight with XIAP (1:200, Cell Signaling), FSCN1 (1:100, Santa Cruz), Ki-67 (1:400, Cell Signaling), Bcl-2 (1:500, Santa Cruz), Bax 1:500, Santa Cruz) and Caspase-3 (1:200, Santa Cruz). Immunostaining was performed using UltraSensitive S-P Detection Kit (KIT-9720, Maixin, Fuzhou, China), and then color was developed by using a DAB kit (DAB-0031, Maixin, Fuzhou, China). Subsequently, sections were counterstained with hematoxylin. TUNEL assay was performed with an in situ cell death detection kit (Roche) according to the manufacturer's instructions. Quantification of immunohistochemical stain intensity was performed as previously described [7 (link), 43 (link)].