FIGURE 8.

FBLN5 knockdown potentiates apoptosis under hypoxia. HUVEC were transfected with a FBLN5-specific siRNA (siFBLN5) or a control siRNA (siRAND) and exposed to normoxia or hypoxia during 24 h. Apoptosis was assessed by annexin V/propidium iodide staining and FACS analysis. A, representative FACS analyses corresponding to one experiment are shown. B, graphical representation showing the percentage of apoptotic cells under each experimental condition. Data are reported as mean ± S.E. (n = 9; *, p < 0.05 versus cells transfected with the same siRNA and maintained in normoxia; †, versus cells transfected with siRAND and maintained under the same experimental condition). C, Western blot analysis demonstrate the efficiency of FBLN5 knockdown in both cells maintained under normoxia or exposed to hypoxia. D, representative confocal images showing FBLN5 staining (in green) from cells transfected with either siRAND or siFBLN5 and exposed to hypoxia. Nuclei were stained with Hoechst (blue), and F-actin is visualized in red. Bar, 10 μm. E, HUVEC were transfected with siRAND or siFBLN5 for 48 h and were exposed to hypoxia for the indicated times. The effect of FBLN5 silencing on total Akt (t-Akt) and on the activation of Akt (p-Akt) and ERK1/2 (p-ERK1/2) was evaluated by Western blot. F, mRNA levels corresponding to FBLN2, FBLN3, and FBLN5 in HUVEC transfected with siRAND (white bars) or siFBLN5 (black bars) were analyzed by real-time PCR (n = 9; *, p < 0.05 versus cells transfected with siRAND.).