Five groups of BXPC‐3 cells were taken. Two groups were transfected with REG4 siRNA (3 μL and 6 μL), another was incubated with 1 μg/mL REG4 mAb, and two were used as the blank control and siRNA control. Then BXPC‐3 cells were collected and suspended in serum‐free DMEM at a density of 2.0 × 106 cells/mL. Five samples of PANC‐1 cells were taken. One was suspended in serum‐free DMEM containing 2 μg/mL rREG4, the second suspended in CM, the third suspended in CMAb, the fourth suspended in serum‐free DMEM containing 2 μg/mL rREG4 after being pre‐incubated with 1 μg/mL REG4 mAb, and the fifth was used as control. All of these PANC‐1 cells were suspended at a density of 2.0 × 106 cells/mL. The same procedure was applied for ASPC‐1 cells.
Transwell migration assays were carried out using 24‐well Millicell Hanging Cell Culture inserts with 8 μm PET (Millipore, Boston, MA, USA). The invasion assay was carried out using QCM 24‐well cell invasion assay kit (ECM554; Millipore) pre‐coated with ECMatrix, a reconstituted basement membrane matrix of proteins derived from the Engelbreth–Holm–Swarm mouse tumor. After the RT‐PCR qualification, which suggested a possible correlation between REG4 and MMP‐7, MMP‐9, a Transwell invasion assay was carried out to test the effect of MMP inhibitor on REG4‐induced enhanced invasion. After pre‐incubation with rREG4, PANC‐1 cells were treated with MMP antibody or siRNA, then collected for subsequent Transwell invasion assay.
Cells (2.0 × 105) were seeded in the upper chamber. NIH 3T3‐fibroblast conditioned medium was added to the lower chamber. After 48 h of incubation at 37°C in 5% CO2, cells on the upper surface of the inner chamber were removed. Migrating or invading cells that adhered to the lower surface of the membrane were fixed and stained with H&E. The migrating or invading cells were counted at ×400 magnification in 10 different fields for each insert. Full paper
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