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. Author manuscript; available in PMC: 2010 Apr 1.
Published in final edited form as: Stroke. 2009 Feb 26;40(4):1474–1481. doi: 10.1161/STROKEAHA.108.527135

Figure 4. Serum-deprivation of endothelial cells results in increased CCM3, activated caspase 3 and p38 levels.

Figure 4

a. Serum-deprived HUVECs demonstrate an increase in cleaved caspase 3 levels starting at 3 hrs post serum withdrawal (global ANOVA P<10-3). At different time points following serum withdrawal, cells lysates were prepared, and the cleaved caspase 3 levels were detected with Western blotting. Graphed densitometric analysis of cleaved caspase 3 is normalized to GAPDH. *corrected P < 0.05 compared with the group in 20% FBS at 0 hour (n = 3).

b. CCM3 expression increases upon serum-deprivation starting at approximately 30 minutes, preceding the increase in cleaved caspase-3 levels as above (global ANOVA P <10-5). * pair-wise corrected P < 0.01 compared with the group at 0 hour (n = 3).

c. Addition of 20% FBS to the cell cultures reduces the increase of CCM3 expression elicited by serum deprivation. Cells were cultured under 0.2% FBS for 1 hour, then exposed to 20% FBS or remained in 0.2% FBS, as described in the Figure. The graphed densitometric analysis of cleaved caspase 3 is normalized to GAPDH as demonstrated by the Western blot on top. *P < 0.05 compared between the 0.2% FBS treated group and the 20% FBS-treated group at 8 and 24 hour time points (n = 3).

d. Actinomycin D treatment of serum-deprived HUVECs prevents the increase in CCM3 protein levels, indicating that serum deprivation causes an increase in CCM3 transcription. HUVECs were incubated with various concentrations of actinomycin D, a transcription inhibitor, for 4 hours in 20% FBS, then exposed to 0.2% FBS still containing various concentrations of actinomycin D; cell lysates were prepared after 1 hour, and CCM3 levels were investigated with Western blotting. *corrected P < 0.01 compared with the group at 0 hour (n = 3).

e. Serum deprivation results in increase in phosphorylation of p38, again preceding the increase in cleaved caspase 3 levels [see a.] (global ANOVA P <10-5). p38 phosphorylation increases dramatically upon serum starvation at 5 minutes, followed by a second peak beginning at 3 hours post serum withdrawal. *corrected P < 0.05 compared with non-0.2% FBS-treated group at 0 hour (n = 3).