TCM was then stored in aliquots at −80°C until used. Each corresponding CP-690550 concentration well was subsequently trypsinized and the number of live cells was counted to allow appropriate correction of TCM loading for cell equivalents. For the MMP-2 blocking assay, TCM was preincubated with MMP-2-neutralizing antibody (#MS-567-P1ABX, Thermo Scientific, Braunsweig, Germany) or the isotype-matched control immunoglobulin G (IgG) (MAB002, R&D Systems, Minneapolis, MN) for 1 hour at 37°C, before being applied to coculture with HUVECs. HUVECs (1.5 × 104) were grown in the absence or presence of 75% TCM for 10 hours at 37°C in a

96-well plate coated with Matrigel (3432-005-01, R&D Systems). The formation of capillary-like structures was captured under a light microscope. The branch points of the formed tubes, which represent the degree of angiogenesis in vitro, were scanned and quantitated in five low-power fields (100×). The 24-well Boyden chamber with 8-μm pore size polycarbonate membrane (Corning, NY) was used to analyze the migration and invasion of tumor cells. For invasion assay, the membrane was coated see more with

Matrigel to form a matrix barrier. Wound healing assay was applied to examine the migration of HUVECs. Details are in the Supporting Materials and Methods. The proliferation of HUVECs was assessed by bromodeoxyuridine (BrdU) incorporation assay, as described in the Supporting Materials and Methods. All experimental procedures involving animals were performed in accordance with the Guide for the Care and Use of Laboratory Animals (NIH publications Nos. 80-23, revised 1996), and according to the this website institutional ethical guidelines for animal experiments. For subcutaneous xenograft model, LM6 cells (1 × 106) that were transiently transfected with miR-29b or NC duplex were

suspended in 100 μL 1 × PBS and then injected subcutaneously into either side of the posterior flank of the same female BALB/c athymic nude mice at 5 weeks of age. Five nude mice were included and tumor growth was examined over the course of 35 days. For orthotopic liver xenograft model, 3 × 106 LM6-miR-29b or LM6-vec cells were suspended in 40 μL of PBS/Matrigel (1:1) and then inoculated under the capsule of the left hepatic lobe of BALB/c nude mice. miR-29b expression was silenced by administering drinking water supplemented with 10% sucrose plus 2 mg/mL doxcycline (Dox, ClonTech). The animals were sacrificed and tumors or livers were dissected, fixed in formalin, and embedded in paraffin. To evaluate intrahepatic metastasis, serial sections of liver were screened. Growth-factor-reduced Matrigel (500 μL, cat. 3433-005-01, R&D Systems) premixed with 2 × 106 LM6-miR-29b or LM6-vec cells was subcutaneously implanted into either side of the flank of the same BALB/c nude mice for 7 days, Matrigel plugs were then dissected, embedded in OCT (Miles, Elkhart, IN), and stored at −80°C. HEK293T cells grown in a 48-well plate were cotransfected with 200 ng of either pcDNA3.