Transwell chamber

The transwell chambers (8‐μm pore size, Millipore) were coated with matrigel (BD Biosciences) diluted with RPMI‐1640 (1:5) on the upper chamber in advance. The cells (A549‐Control, A549‐SELENBP1, H1299‐Control, and H1299‐SELENBP1) were seeded into the transwell chambers (8‐μm pore size, Merck, Millipore) with serum‐free media and each transwell chamber contained around 4 × 10⁵ cells. Media containing 10% FBS was added to the lower chamber, cultured at 37°C incubator for 24 h, and then fixed in 4% paraformaldehyde; the noninvading cells on the upper of the chambers were removed with cotton wool; invasive cells located on the lower surface of the chamber were stained by crystal violet stain (Sigma Diagnostics). To count the invasive cells, pictures were taken using a Zeiss Imager Z2 microscope (Carl Zeiss; 200×).

MCF-7 breast cancer cell strains and 293T cells were purchased from the Shanghai Cell Resource Center of the Chinese Academy of Sciences (Shanghai, China). The pSPAX2, pMD2G and pLVX-shRNA1 vectors were purchased from Clontech Laboratories (Mountain View, CA, USA). The Plasmid Midi kit was purchased from Qiagen (Valencia, CA, USA). Opi-MEM, Escherichia coli DH5α and Taq DNA polymerase were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). T4 DNA ligase, BamHI and EcoRI restriction enzymes were purchased from New England Biolabs (Ipswich, MA, USA). Liposome Lipfectamine 2000, Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS) and Trypsin were purchased from Invitrogen Life Technologies. The gel extraction kit was purchased from Tiangen Biotech (Beijing) Co., Ltd. (Beijing, Japan). KOD high fidelity enzyme polymerase chain reaction (PCR) kit and Taq enzymes were purchased from Toyobo Co., Ltd. (Osaka, Japan). DNA ladder was purchased from Fermentas International, Inc., (Burlington, Canada). The Transwell chamber was purchased from Chemicon (Temecula, CA, USA).

Transwell chamber ([8.0 μm pore size; EMD Millipore]) with (or without) Matrigel (Becton Dickinson) was applied for the assessment of invasion (or migration) of LUSC cells. The cells in serum‐free medium were placed in the upper chamber, while the lower chamber was filled with medium supplemented with 10% bovine calf serum. Methanol was used to fix the cells after 48 hours, and crystal violet (0.1%) was employed to stain the cells. Finally, the cells that had migrated or invaded were examined under the microscope.

To determine whether the recombinant S100A4 protein stimulated cell migration and invasion, cell motility and invasion assays were conducted in a Transwell chamber (EMD Millipore) (11 (link)), which was coated with Matrigel^®. Assays were conducted according to the manufacturer’s instructions. Following overnight starvation in RPMI-1640 media and the addition of a medium containing 10% FBS to the bottom chambers as a chemoattractant, HeLa cells (1×10⁵) were added to the top chambers of the 24-well transwell plates as the control group (C), whereas HeLa cells (1×10⁵) with recombinant human S100A4 were added into another top chamber as the experimental group (E). The cells were then incubated for 24 h at 37°C. Non-motile cells on top of each chamber were removed by wiping with cotton swabs. The cells on the bottom of each chamber were fixed with 0.1% glutaraldehyde for 30 min, rinsed briefly with phosphate-buffered saline (PBS) and stained with 0.2% crystal violet (Ameresco, Inc., Framingham, MA, USA) for 20 min. The chambers were then washed thoroughly with PBS. The number of migrated or invaded cells was calculated under ×200 magnification and the mean for each chamber was determined. The results were calculated as the migration/invasion rate as compared with the parental control cells. Each experimental condition was conducted in duplicate and repeated at least three times.

A Transwell chamber (EMD Millipore, Billerica, MA, USA) was used for invasion assessment of T24 cells. Then, 2 × 10⁵ T24 cells were placed into the top of the insert precoated with Matrigel (BD Biosciences, USA) and the medium+10% FBS was added into the lower chamber. After being maintained in a moist incubator under routine conditions for 24 h, cells that failed to invade were discarded, while cells that invade to the other side were fixed 10 min with 4% paraformaldehyde and stained 30 min with crystal violet. Five fields in each well were randomly selected after drying, and cells were counted under an inverted microscope.