Cell Line and Cell Culture The human colon cancer cell line HCT116 was purchased

from China Centre for Type Culture Collection. The cells were grown in McCoy’s 5A medium, Modified (Sigma), supplemented with 10% of fetal bovine serum (Hyclone, USA) at 37°C in a humidified atmosphere of 5% CO2. The cells were always detached using 0.25% trypsin and 0.02% ethylene diamine tetra acetic acid(EDTA). In vivo Tumor Xenograft Model To PD-1/PD-L1 Inhibitor 3 cell line establish the transplantable model, the human colon cancer cells in logarithm growth phrase were harvested and washed twice with PBS. 1.0 × 107 cells in 200 uL of PBS with a viability of >95% tested by staining with trypan blue were injected subcutaneously into the right flank of each mouse. All nude mice were observed to generate tumors for up to 9 days after the injection. When tumor nodules reached 5-7 mm in diameter, tumor model was successfully established and mice were randomly assigned to the following 3 groups(seven this website mice in each group): (1)normal saline(NS)

group, (2) Ad-HK group and (3) Ad-RhoA-RhoC group. Ad-HK (4 × 108 pfu, 30 ul/mouse), Ad-RhoA-RhoC (4 × 108 pfu, 30 ul/mouse) or PBS (30 ul/mouse) was injected intratumorally at several points four times once every other day, with the accumulated doses of 1.6 × 109 pfu. The tumor sizes were determined every other day by external measurements

with a vernier caliper and calculated the tumor volume and plotted against time [The tumor volume = ab2/2, where a and b are the larger and smaller diameter, respectively]. Ten days after the final injection, the tumors were dissected and their weights and volumes were measured. Then, each harvested tumor was divided into two parts, one was used for detecting the mRNA expression of the related genes and the other was used for immunohistochemical analysis as described below. Quantitative RT-PCR for RhoA and RhoC in Xenograft Tumors Total RNA was extracted from Decitabine solubility dmso -80°C freezed transplanted tumor samples, dissected from nude mice, using Trizol reagent(click here Invitrogen, USA) and reverse transcripted into cDNA using the PrimeScript RT-PCR kit (TaKaRa Bio Inc., Shiga, Japan), according to the manufacturer’s instructions. To assess the RhoA and RhoC gene expression, we used real-time fluorescence quantitative PCR analysis based on the TaqMan probe method. The probe contains 6-carboxy-fluorescein (FAM) as a fluorescent reporter dye, and 6-carboxytetramethyl-rhodamine (TAMRA) as a quencher for its emission spectrum. The primers, TaqMan probes and PCR parameters were performed same as reported previously by us [18, 19].