The culture media were changed once per 48 h. The Afatinib ic50 lowest G418 concentration, in which all cell died after 12-14 days culture, was chosen as the optimal concentration for resistance

selection. Transfection of SHG44 cells with pcDNA3.1-DKK-1 For stable transfection of the DKK-1 gene, SHG44 cells (1 × 106) were plated in 6-well plates 24 h before transfection. Lipofectamine 2000 (Invitrogen Company) was used to mediate transfection using 5.0 μg of pcDNA3.1-DKK-1 vector or 5.0 μg of empty pcDNA3.1 vector as a control according to the manufacture’s protocol. After 48 h transfection, the cells were selected in media supplemented with G418 (150 μg/ml). The medium was changed once per 48 h. Non-transfected SHG44 cells died within two weeks. G418-resistant cells were selected and named as SHG44-DKK-1. Cells with empty vector of pcDNA3.1 were named as SHG44-EV. PCR confirmation of DKK-1 in SHG44 cells DNA from cells of normal SHG44, SHG44 -EV, SHG44-DKK-1 was isolated using a DNA extraction kit (Puregenetm DNA isolation kit, Gentra systems). Metformin datasheet A portion of the DKK-1 gene was used to design the primers. The upstream primer sequence was 5′-TCACGCTATGTGCTGCCCCG-3′ and downstream 5′-TGAGGCACAGTCTGATGACCGGA-3′. The expected product was 223 bp. PCR reaction system

(50 μl) was: 3 μl cDNA, 5 μl 10 × Buffer, 4 μl MgC12, 1 μl dNTP, 1 μl primer, 0.3 μl TaqDNA Polymerase. PCR reaction condition was: an initial denaturation step of 94°C for 7 min, followed by 30 cycles of a three-step program of 94°C for 30 s, 56°C for 30 s, 72°C for 45 s, and a final extension step of 72°C for 7 min. All the products were electrophoresed on the agarose gel. RT-PCR of DKK-1 mRNA Analysis of the DKK-1 mRNA expression of the three groups of cells (normal SHG44, SHG44-EV and SHG44-DKK-1) was performed by RT-PCR. Total RNA from cell lines was isolated using Trizol (Invitrogen Company). The purity and concentration of total RNA were detected by UV chromatogram analyzer (Backma Company). The concentration Cyclooxygenase (COX) of RNA was adjusted to 1 μg/μl. β-actin

was used as an internal control to ensure RNA quality and loading accuracy. Primer sequences were 5′-AGCGAGCATCCCCCA AAGTT-3′ (upstream) and 5′-GGGCACGAA GGCTCATCATT-3′ (downstream). The predicted product size is 285 bp. The primers for DKK-1 were the same mentioned above. The PCR condition for DKK-1 and β-actin was the same as described above. Western blot analysis The total protein of the three groups of cells (normal SHG44, SHG44-EV, SHG44-DKK-1) was extracted directly in the lysis buffer and the concentration of total protein was quantified by UV chromatogram analyzer. 50 μg protein was separated using 12% sodium dodecyl sulfate- polyacrylamide gel (SDS-PAGE). After electrophoresis, proteins were transferred from gel to zapon fibrous membrane and the membrane was blocked by 5% non-fat milk. Monoclonal mouse anti-human DKK-1 antibody (R & D Company) (1:1000 dilution) was probed.