PCR was
performed using cDNA PCR kits (Takara, Cat. DRR019A, Japan) in a final volume of 50 μl according to the manufacturer’s instructions. Amplification conditions were performed for 30 cycles (denaturation at 94°C for 1 min, annealing at 54°C for 1 min, and extension at 72°C for 1 min). The MMP7 primers see more were 5′-AGA TGT GGA GTG CCA GAT GT-3′ (find more forward) and 5′-TAG ACT GCT ACC ATC CGT CC-3′ (reverse). The ERβ primers were 5′-TGC TTT GGT TTG GGT GAT TGC-3′ (forward) and 5′-TTT GCT TTT ACT GTC CTC TGC-3′ (reverse). The β-actin primers were 5′-CGG GAC CTG ACT GAC TAC CTC A-3′ (forward) and 5′-TCA AGA AAG GGT GTA ACG CAA CTA-3′ (reverse). The PCR products were separated selleck by electrophoresis on a 2% agarose gel and visualized by ethidium bromide staining and UV illumination. The expected sizes of the amplification products were 365 base pairs (bp) for MMP7, 259 bp for ERβ, and 656 bp for β-actin. Western blotting HT-29 cells were exposed to TAM, 5-FU, or their combinations for various time points in various administration sequences. After treatment, 5 × 106 cells were collected for protein extraction. Cell pellets were washed in PBS twice and then lysed in 80 μl lysis buffer (0.1% SDS, 50 mmol/L Tris·HCl pH 8.0, 150 mmol/L NaCl, 1 mmol/L EDTA, 100 μg/ml PMSF, 1 μg/ml Aprotinin, 1% NP-40) for 30 min on ice. After centrifugation
at 12,000 rpm for 5 min at 4°C, the supernatants were collected and frozen at -80°C until analysis. Forty micrograms of total protein were loaded in each well of a 10% SDS-PAGE gel. Proteins were transferred to Hybond P polyvinylidene fluoride membranes (Amersham Pharmacia Biotech, Amersham, UK), which were then blocked in 5% dried skimmed milk powder in TBST (Tween 20/TBS) for 3 h at room temperature. Membranes were probed with primary antibodies (mouse monoclonal MMP7 and ERβ antibody, 1/1000) and
then horseradish peroxidase-conjungated second antibody. After washing, the immunoreactive protein was detected using chemiluminescence (Cell Signaling). Wound scratch assay HT29 cells (2 × 105) were cultured to confluent cell monolayers in medium containing 10% FBS on 6-well tissue culture dishes. Cells were carefully wounded using sterile 20-μl pipette tips. The wounded monolayers Lenvatinib in vivo were washed twice with PBS to remove nonadherent cells and incubated at 37°C in complete media. The cells were then incubated in TAM (according to the drug administration schedule) for 24 h, 48 h, or 72 h. The wound edges were imaged by phase-contrast microscopy, and the extent of migration was analyzed using the NIH image software http://rsb.info.nih.gov/nih-image/Default.html. Statistical analysis The results are presented as the mean ± SD. P values less than 0.05 were considered statistically significant.