Influence of cell density on modulators of the MAPK signaling pathway in ECs. (A) Western blots for phospho-(p)-ERK1/2 and pan-ERK1/2 from EC lysates. Confluent (20 000), subconfluent (10 000), and sparse (5000) HUVECs were grown for 4 days in complete growth medium and afterward rendered quiescent for 4 hours by serum deprivation (4% bovine serum albumin in M199). Cells were then stimulated with 20 ng/mL VEGF165 for the time indicated. All samples were lysed in same volumes and obtained as described in “Western blotting.” Quantification of pERK1/2 chemiluminescence normalized to pan-ERK1/2 at 10 minutes of VEGF165 stimulation (RLU): 0.36/1 (5000), 0.16/0.38 (10 000), 0.05/0.1 (20 000); ERK1 = p44mapk, ERK2 = p42mapk. (B) Relative quantitative reverse transcriptase-PCR of HUVECs seeded in different densities (indicated as cells per centimeter squared) and grown in complete growth medium for 4 days. Among major phosphatases impacting the MAPK signaling pathway, only DEP-1 mRNA levels increased significantly; values were normalized to porphobilinogen deaminase. Data are mean ± SEM. *P < .05. n.s. indicates not significant.
