5 × 1010 cells/L suspension in serum-free RPMI-1640 medium. 0.2 mL

cell suspension was subcutaneously inoculated in the right armpit of each mouse. 21 days after inoculation, 29 out of 50 mice had tumor volume ≥ 500 mm3 and randomly assigned into 4 groups[6]. MCF-7 cell was innoculated into the other 50 nude mice for building the model[7]. 5. MDA-MB-231 and MCF-7 cell invasion assay Breast cancer cell invasion was measured using Transwell chamber. In detail, 2 × 105 cells were placed in the upper chamber of Transwell with a membrane coated with Matrigel. 24 h later, cells were incubated with 800 U/mL ulinastatin, 3.7 μg/mL docetaxel, 800 U/mL ulinastatin plus 3.7 μg/mL docetaxel, and PBS, respectively, at 37°C in an incubator supplemented with 5% CO2. 24 h later, cells in the upper chamber were removed with GDC-0449 a cotton swab. The remaining cells on the membrane were stained with 0.1% crystal violet solution and washed with PBS. Crystal violet attached to the cells was dissolved by adding 500 μL of 33% acetic acid into the lower chamber and its absorbance at 570 nm was measured and

used to calculate relative amount of cells invaded through the Matrigel to the lower chamber. 6. mRNA levels of uPA, uPAR and ERK in MDA-MB-231 and MCF-7 cells measured by real-time RT-PCR To evaluate the effect of treatments described above on mRNA levels of uPA, uPAR and ERK in breast cancer cells, 24 h after the treatment, total mRNAs were isolated using 1 mL TRIzol reagent according to the protocol provided by the manufacturer. 20 μL mRNA was reverse transcripted into cDNA CX5461 and the amount of uPA, uPAR and ERK cDNA was examined by quantitative real-time PCR using the following primer pairs: uPA Protein kinase N1 forward primer 5′-GGAGATGAAGTTTGAGGT-GG-3′ and reverse primer 5′-GGTCTGTATAGTCCGGG-ATG-3′, uPAR forward primer

5′-CACAAAACTGCCTCCTTCCT-3′ and reverse primer 5′-AATCCCCGTTGGTCTTACAC-3′, ERK forward primer 5′-CCTAAGGAAAAG-CTCAAAGA-3′ and reverse primer 5′-AAAGTGGATAA-GCCAAGAC-3′, and β-actin forward primer 5′-GCAGAAGGAGATCACAGCCCT-3′ and reverse primer 5′-GCTGATCCACATCTGCTGGAA-3′. The corresponding predicted products were 142, 178, 180, and 136 bp, respectively. In detail, template cDNA and HSP activation primers were mixed with SYBR Green/ROX qPCR Master Mix (2X) in 25 μL reaction system and PCR was carried out in triplicate under the following conditions: 5 min at 95°C, 45 cycles of 15 seconds at 95°C and 30 seconds at 60°C, 1 min at 95°C and 1 minute at 55°C. Ct value of each sample was defined as cycle number when the fluorescence intensity reached the threshold. Relative RNA level was normalized to β-actin and quantified using 2-ΔΔ. 7. Protein expression of uPA, uPAR and p-ERK1/2 determined by Western blot 24 h after treated as described above, MDA-MB-231 cells were lysed with 25 μL buffer and mixed with 2× sample buffer. Proteins were then subjected to SDS-PAGE and transferred onto PVDF membrane.