Microphot fx

After 24 h transfection, cell concentration was adjusted to 5 × 10⁴ cells/ml with serum-free medium. The upper chamber of Transwell chamber (Costar; 24-well insert, pore size: 8 μ m) was filled with 200 μl of cell suspension, and the lower chamber was filled with 500 μl of medium with 10% FBS. For the invasion assay, polycarbonate filters coated with 50 μl Matrigel (1:9, BD Bioscience) were placed in a Transwell chamber. Cells were incubated for 12 h for the migration assay, and 36 h for the invasion assay. Cells on the bottom surface of the membrane were fixed with 4% paraformaldehyde and stained with 0.5 % crystal violet. The migratory cells were visualized and counted in five random visual fields per insert under an inverted microscope at 100× magnification (Nikon MicrophotFX, Japan). The experiment was repeated three times.

After 48 h of transfection, cell concentration in each group was adjusted to 2 × 10⁵ cells/mL with serum-free medium. The upper chamber of Transwell chamber (Costar; 24-well insert, pore size: 8 μm) was filled with 200 μl cell suspension, and the lower chamber was filled with 500 μL of medium supplementing 15 % FBS. For the invasion assay, polycarbonate filters coated with 50 μL Matrigel (1:9, BD Bioscience) were placed in a Transwell chamber. Three wells were used for each group. Cells were incubated for 24 h for the migration assay and 48 h for the invasion assay. Then, the cells on the upper surface were wiped slightly using cotton swabs, and the cells on the lower surface were fixed with 4 % paraformaldehyde and stained with 0.1 % crystal violet. The migratory cells were visualized and counted in five random visual fields per insert under an inverted microscope at 200× magnification (Nikon Microphot-FX, Japan).

Matrigel-coated and -uncoated modified Boyden chambers (BD Biosciences, U.S.A.) were used to evaluate cell invasion and migration. Briefly, transfected cells suspended in FBS-free growth medium were plated into the upper chamber, and growth medium containing 10% FBS was loaded in the lower chamber as an attractant. After 24-h incubation, cells were fixed in 4% paraformaldehyde and stained with 0.1% Crystal Violet (Sigma, U.S.A.). The cells on the upper chamber, which had invaded or migrated to the lower chamber, were removed with a cotton swab and were counted by using a microscope (Nikon Microphot-FX, Japan) in five randomly selected field at ×20 magnification.

Cell invasion and migration were detected with modified Boyden chamber (BD Biosciences, Sparks, MD, USA) assays. Briefly, three PC cell lines were seeded onto 8.0-µM pore size membrane inserts with 8.0 µM pores coated with matrigel (BD Biosciences) in 24-well plates with FBS-free growth media. 1640 plus 10% FBS was added to the bottom of the wells as a chemoattractant. 12-24 hrs later, cells that did not migrate were removed from the top side of the inserts with a cotton swab. Cells that had migrated to the underside of the inserts were stained with Crystal Violet Hydrate (Sigma) according to the manufacturer's instruction. The migratory cells were counted under a microscope at 20 x magnification. Cell images were obtained using a microscope (Nikon Microphot-FX, Japan), and counted in five random fields per insert. The migration assay was done in a similar fashion without matrigel. In more detail, we first used the same cell intensity (5×10^5/ml) to investigate different invasion ability of three normal PC cell lines. Because too high or too low cell population in invasion and migration assay is unfavorable for late result analysis in our pre-experiment, we seeded 1×10^5/ml of PANC-1 cells, 2.5×10^5/ml of BxPC-3 cells and 5×10^5/ml of Capan-2 cells in cell invasion and migration assayt. The results are presented as cells (actual number) migrated per field.

PC cells were plated into 24-well culture plates, fixed in 4% paraformal dehyde, permeabilized with 0.25% Triton X-100 and incubated with 5% BSA. Then the slices were stained with the primary antibodies: MSI2 (Abcam, Cambridge, UK), ZEB1 (Proteintech, Chicago, IL, USA), pERK (Cell signaling technology, MA, USA) and c-Myc (Proteintech). The secondary antibodies were conjugated with FITC and TRITC (Vector laboratories, California, USA). Cells were then co-stained with blue Hoechest33258 (Vector laboratories) for nuclear visualizing and detected under microscope (Nikon Microphot-FX, Japan).

The number of TH⁺ neurons in the SN was counted using a stereology system (Microphot-FX, Nikon Corporation, Tokyo, Japan) with a 40x lens. Counting was performed with the slides encrypted and the person counting blinded to the experimental conditions. Cells were counted if the nucleus was present in the counting frame completely or touching the upper and/or right frame lines. Cells touching the lower and/or left frame line were not counted. The SN was outlined as on every 5th serial slice (2.4 to 4.1 mm dorsal of the bregma) and the total number of TH⁺ cells in the SN was determined with the Stereo Investigator software (MBF Bioscience, Williston, VT, USA) using the optical fractionator method.