Apoptosis was determinate LOXO-101 by staining cells with annexin V-FITC and propidium-iodide (PI) labeling, because annexin V can identify the externalization of phosphatidylserine during the apoptotic progression and therefore detect early apoptotic cells [29]. Cells were transduced with TG 9344 vector, on 12-well plates and treated after 24 hr by 20 μM GCV. Control cells were no transduced or untreated. After 72 hr of treatment, cells were harvested, and washed twice in PBS. The pellet was resuspended in 1 ml of 100 mM HEPES/NaOH, pH 7.5.

Then 500 μl of the cell suspension were incubated in presence of 2 μg/ml annexin V-FITC, and 10 μl of PI (100 μg/ml) for 10 min. Samples were MLN2238 cost immediately analyzed by flow cytometry on a bi-parametric histogram giving the level of annexin V-FITC and PI fluorescence. buy BI 6727 Apoptosis was assessed by DNA fragmentation assay. Samples of 5.105 pTG 9344 transduced cells with or without synchronization were treated 96 hr with 20 μM GCV. Cells then were centrifuged at 800 g for 5 min at 4°C. The pellet was resuspended in 20 μl of lysis buffer (EDTA 20 mM, Tris 100 mM, SDS 0,8%,

pH 8). Then 10 μl of 500 UI/ml RNAse (Sigma) were added for 60 min at 37°C. The mix was incubated 90 min at 50°C with 10 μl of 20 mg/ml proteinase K. Migration was achieved on 1.8% agarose gel containing 0.5 μg/ml ethidium bromide at 35 V during 4 hr. MSP I digested PBR 322 was used as a size marker. Non-transduced cells treated with MTX or GCV constituted control groups. Statistical analysis Comparisons were made using the Student’s t test. P < .05 was considered as significant. Results

Altered progression in the cell cycle by methotrexate, ara-C or aphidicolin Lepirudin We first assessed the effect of drugs on DHDK12 and HT29 cell cycles to delineate the time for which a maximum of cells were in S phase after drug removal. The effects of the three drugs, i.e. MTX, ara-C and aphidicolin, on the cell cycle were preliminary assessed in DHDK12 cells. After a 24 hr treatment with MTX, ara-C or aphidicolin, cells were analyzed between 0 and 72 hr after drug removal for DNA content by flow cytometry. In the DHDK12 cell line, 20% of cells were in S phase in the absence of drug and this rate was constant over time (Figure 1A). When DHDK12 cells were treated with ara-C or aphidicolin, 25% and 35% of cells were in S phase 10 hr after ara-C or aphidicolin removal, respectively (Additional file 1). By contrast, treatment with MTX resulted in 51% of the cells to be in S phase, while 28% were in G0-G1 phase, 10 hr after drug removal (Figure 1A). The ratio of cells in S phase remained higher than that in G1 phase up to 30 hr following MTX removal.