One million T cells were fixed with 70% cold ethanol Dibutyryl-cAMP at 4°C for 30 min, washed with PBS twice, and stained with 50 g/ml PI (Sigma, USA) at room temperature for 5 min. Data were analyzed with Mod-FIT software. Effect of MSCs on T cell activation MSCs and MNCs were prepared as described before, respectively. T cells were cultured alone or cocultured with

prepared MSCs and stimulated with PHA (50 g/ml final concentration). The expression of CD25 (BD, USA) and CD69 (BD, USA) was detected by flow cytometry at 24 h, and CD44 (BD, USA) was detected at 72 h. Effect of MSCs on T cell apoptosis MSCs and MNCs were prepared as described before. T cells were cultured alone or cocultured withMSCs with PHA (50 g/ml final concentration) stimulation for 3 days, then harvested and quantified, stained with Annexin-V kit (BD, USA), and analyzed by flow cytometry (FACS Vantage). RNA-i experiments The si-RNA sequence targeting human MMP-9 (from mRNA sequence; Invitrogen online) corresponds to the coding region 377-403 relative to the first

nucleotide of the start codon (target = 5′-AAC ATC ACC TAT TGG ATC CAA ACT AC-3′). Computer analysis using the software developed by Ambion Inc. confirmed this sequence to be a good target. si-RNAs were 21 nucleotides long with symmetric 2-nucleotide 3′overhangs composed of 2′-deoxythymidine to enhance nuclease resistance. The si-RNAs PX-478 datasheet were synthesized chemically and high pressure liquid chromatography purified (Genset, Paris, France). Sense si-RNA sequence was 5′-CAU CAC CUA UUG GAU CCA AdT dT-3′. Antisense si-RNA was 5′-UUG GAU CCA AUA GGU GAU GdT dT-3′. For annealing of si-RNAs, mixture of complementary single stranded RNAs (at equimolar concentration) was incubated in annealing buffer (20 mM Tris-HCl pH 7.5, 50 mM NaCl, and 10 mM MgCl2) for 2 minutes at 95°C followed by a slow cooling to room temperature (at least 25°C) and then proceeded to storage temperature of 4°C. Before transfection, cells cultured at

50% confluence in 6-well plates (10 cm2) were washed two times with OPTIMEM 1 (Invitrogen) without FCS and incubated in 1.5 ml of this medium without FCS for 1 hour. Then, cells were transfected Megestrol Acetate with MMP-9-RNA duplex formulated into Mirus TransIT-TKO transfection reagent (Mirus Corp, Interchim, France) according to the manufacturer’s instructions. Unless otherwise described, transfection used 20 nM RNA duplex in 0.5 ml of transfection medium OPTIMEM 1 without FCS per 5 × 105 cells for 6 hours and then the medium volume was adjusted to 1.5 ml per well with RPMI 2% FCS. SilencerTM negative CFTR inhibitor control 1 si-RNA (Ambion Inc.) was used as negative control under similar conditions (20 nM). The efficiency of silencing is 80% in our assay. Enzyme-linked Immunoadsorbent Assays This was carried out according to the manufacturer’s recommendations (Oncogene Research Products).