RNA purification and reverse transcriptase polymerase chain reaction Total RNA from homogenized bulky tissues and cell lines was extracted using RNeasy Mini Kit, and reverse transcribed with a SuperScript III Reverse Transcriptase the kit. Quantitative real time RT PCR for HOPX B or HOPX core was performed using iQTM SYBR Green Supermix in triplicate on the iCycler iQTM Real Time PCR Detec tion system, either. Relative quantita tive analysis adjusted for B actin was performed according to the CT method. Table 1 depicts sequences of primers/probes and PCR condition. Immunoprecipitation and Western blotting Whole cells lysates were obtained using RIPA buffer supplemented with 10 uL/ ml HaltTM Protease Inhibitor Cocktail Kit and HaltTM Phosphatase Inhibitor Cocktail Kit.

Immunoprecipitation was performed using Dyna beads Protein G, 1 ug of anti HOPX mouse IgG1K monoclonal antibody, and 400 ug of each cell lysates. The anti HOPX mouse IgG1K monoclonal antibody, anti HOPX rabbit IgG polyclonal anti body, anti V5 mouse IgG2a monoclonal antibody, and anti B actin mouse IgG2a monoclonal antibody were used for Western blotting or IP/WB. 5 Aza dC and TSA treatment Cells were treated with 1 or 5 uM of the demethylating agent 5 aza 20 deoxycytidine dissolved in 50% acetic acid or mock treatment with PBS including the same amount of acetic acid every 24 hrs for 4 days. When combined with the histone deacetylase inhibitor trichostatin A, 300 nM TSA was added to the medium for the final 24 hrs. Immunohistochemistry Formalin fixed, paraffin embedded histological sections were immunostained using the HOPX antibody.

And immune com plexes were detected using the 3,30 diamino benzidine tetrahydrochloride substrate, as a chromogen for 30 seconds or 2 minutes. Plasmid and transfection A full length cDNA of HOPX was previously isolated and subcloned into pcDNATM3. 1D/V5 His TOPO vec tor. The vector with self ligation was used as a mock control. Plasmid vectors were transfected into 2 pancreatic cancer cell lines using Lipofectamine 2000 reagent. Stable clones with HOPX or mock were established by G418 selection. Proliferation assay Cell proliferation and viability were measured using the Premix WST 1 Cell Proliferation Assay System in 96 well plates. Experiments were performed in triplicate. Invasion assay Cells were seeded at density of 1�� 106 cells/well in the 24 well BD BioCoat Matrigel Invasion Chamber filled with 500 ul DMEM.

As a chemoattractant, 10% FBS in 750 ul DMEM was used for the assay. After incubation for 22 hrs, the membrane of the upper cham ber was Anacetrapib fixed and stained by Diff Quick reagent. Invaded cells were counted in for randomly selected sites per membrane. Anchorage independent colony formation assay Anchorage independent cell growth was analyzed by plating 0. 36% top agarose containing 1��105 cells on a surface of 0. 72% bottom agarose in 6 well plates.