FIGURE 1.
Hypoxia increases FBLN5 expression. BAEC were maintained under normoxic conditions (white bars), exposed to hypoxia (1% O2; black bars) during different times or treated with dimethyl oxal glycine (0.5 mm, 24 h; gray bar). A, FBLN5 mRNA levels were evaluated by real-time PCR and were normalized by TATA-binding protein expression levels. Data are expressed as mean ± S.E. (n = 9; *, p < 0.05 versus control cells). B, HIF-1α protein levels were evaluated in cell lysates by Western blot. Unchanged levels of β-actin are shown as a loading control (Norm, normoxia). Autoradiograms are representative of two independent experiments performed in duplicate. C and D, FBLN5 mRNA levels were analyzed in HUVEC (C) and HeLa (D) exposed to hypoxia for 24 h. Data are expressed as mean ± S.E. (n = 9; *, p < 0.05 versus control cells). E, HUVEC and HeLa were maintained under normoxia or exposed to hypoxia for 48 h. FBLN5 protein levels were analyzed by Western blot from whole-cell extracts or supernatant from cell cultures. β-Actin levels were used as a loading control. A representative autoradiogram of three independent experiments performed by duplicate is shown. F, FBLN5 localization was analyzed by immunocytochemistry in HUVEC maintained in normoxia (a and c) or exposed to hypoxia (b and d). In permeabilized cells, intracellular FBLN5 staining (green) was detected in the perinuclear region and in cell periphery (a and b). Extracellular localization was analyzed in nonpermeabilized cells (c and d). Cells were counterstained with Hoechst to highlight nuclei (blue) and with Alexa Fluor 633-phalloidin to visualize F-actin (red). Bar, 10 μm.