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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 5. siRNA inhibition of CCM3 expression in endothelial cells results in decreased cell death, p38, and cleaved caspase-3 expression.

Figure 5

a. Serum-deprived HUVECs treated with CCM3 siRNA show decreased cell death. Representative images (20x original magnification) at the 3-hour time point are shown for untransfected HUVECs (no siRNA group, left panel) or those transfected either with control siRNA (middle panel) or CCM3 siRNA (right panel). All groups were subjected to serum deprivation (see Methods) and cell death was analyzed at various time points by TUNEL assay. TUNEL-positive cells (green) are scarce in the sample treated with CCM3 siRNA (right panel) compared to those untreated (left panel) or treated with control siRNA (middle panel).

b. The percentage of apoptotic cells was calculated from TUNEL positive cells in each field (400x) and in at least 5 fields for each group (global ANOVA P <10-3 in both control groups across all time points and within the 3 hour group). *corrected P < 0.05 compared with the equivalent groups prior to serum deprivation (0 hour) (n = 3).

c. Serum-deprived HUVECs show decreased cleaved caspase 3 levels after treatment with CCM3 siRNA (global ANOVA P < 10-3 in both control groups across two time points). HUVECs were transfected with control siRNA or CCM3 siRNA, then were cultured under serum deprivation conditions (0.2% FBS). Cells lysates were prepared at various time points and cleaved caspase 3 levels were analyzed using Western blotting. Serum deprivation results in increase of cleaved caspase 3 in both the no siRNA and control siRNA treated groups, but not in the CCM3 siRNA-treated group. *corrected P < 0.05 compared with the equivalent groups prior to serum deprivation (0 hour) (n = 3).

d. HUVECs treated with CCM3 siRNA demonstrate decreased p38 activation (global ANOVA P < 10-3 in p-p38/p38 and CCM3/GSDPH groups across 2 time points). Similar to the procedure in [c], serum deprivation results in increase of p38 activation and CCM3 protein levels in the no siRNA- and control siRNA-treated groups, but not in the CCM3 siRNA-treated group. *corrected P < 0.05 compared with the equivalent groups at each time point (n = 3).