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. 2006 Nov 6;103(46):17260–17265. doi: 10.1073/pnas.0605556103

Fig. 3.

Fig. 3.

VEGF specifically mediates TGF-β1 induction of apoptosis with a mechanism independent of its proliferative activity. (A) BrdU uptake (% positive cells) by BCE cells in medium containing 10% FCS (Control 10% serum) or grown overnight in 0.5% FCS and incubated for 6 h without (Control) or with TGF-β1 (1 ng/ml) with or without VEGF antibody (VEGF mAb) or n.i. IgG (10 μg/ml) or with anti-VEGF antibody alone. Means ± SE of a representative experiment are shown. (B) Apoptotic index (%). The same cells were immunostained with antibody to cleaved caspase 3 and counterstained with DAPI. The bars show the percentage of cleaved caspase 3-positive cells per total number of nuclei counted (1,000). Means ± SE of a representative experiment are shown. (C) Western blotting analysis of cleaved caspase 3 in BCE cells incubated without (Control) or with TGF-β1 (1 ng/ml) or TNF-α (10 ng/ml plus 1 μg/ml cycloheximide) with or without anti-VEGF antibody (VEGF mAb) or n.i. IgG (10 μg/ml). (Lower) BCE cells irradiated with UVB (20 mJ/cm2) or treated with H2O2 (400 μM) and incubated for 6 h with the indicated reagents. In B and C, ERK2 was used as a loading control. (D) Immunocytochemical analysis of caspase 3 activation in cells treated as described above with TGF-β1 or TNF-α and the indicated reagents. The apoptotic index was measured as described in B. These experiments were repeated twice with comparable results.