Kinetics of RCAS1 signaling in ECFCs.

Day 7 ECFCs were cultured with (■) or without (░) rhEPO (2 U/mL), or with CH-11 (500 ng/mL; ▨) plus rhEPO, or with nRCAS1 (40μg/mL; ▪) plus rhEPO. Each point shows the mean ± SD of triplicates. (A) Detection of apoptotic cells. Apoptotic cells were determined using annexin V staining at the indicated times. (B) Loss of mitochondrial transmembrane potential (ΔΨm). Cells (1.0 × 10⁵) were incubated with 40 nM 3,3′-dihexyloxacarbocyanine(DiOC₆) for 15 minutes at 37°C, and pelleted by centrifugation. Cells resuspended in 500 μL PBS were analyzed by flow cytometry. (C) Activation of caspase 8. Lysates from 1 × 10⁶ cells were incubated with fluorogenic substrate (IETDAFC) for 60 minutes at 37°C in buffer containing 5 mM dithiotreitol. Samples were then analyzed using a multilabel counter at 535 nm. (D) Activation of caspase 3. Cells (1 × 10⁶) were washed twice with PBS, and 50μL substrate solution (10 mM; GDEVDGI) was added, prior to incubation for 60 minutes in a 5% CO₂, 95% air incubator at 37°C. Ice-cold flow cytometry solution (500 μL) was added to each sample followed by flow cytometry analysis.