Figure 2.

FasL induced BM–MSC apoptosis. (a) BM–MSCs from batch no. 1 were treated with 25 ng/ml FasL (black bars) or left untreated (white bars); the percentage of apoptotic cells was evaluated by Hoechst staining and fluorescence microscopy analysis on days 1, 2, 4, and 6. Data are expressed as mean±S.D. (b) BM–MSCs from batch no. 1 were treated with different FasL doses ranging from 0.5 to 25 ng/ml (black bars) or with 50 and 1000 ng/ml of anti-Fas human activating clone CH11 (white bars). The percentage of apoptotic cells was evaluated on day 1 as in (a). Data are expressed as mean±S.D. **P<0.01. (c) FasL-induced caspase 8 and 3 activation was assayed by western blotting of their pro-enzymes, pro-casp-8, and pro-casp-3 (mouse anti-caspase 3, no. 9668) on day 1 of treatment. The pan caspase inhibitor zVAD (20 μℳ) was added 20 min before treatment to control the specificity of activation. N=3. One representative western blot is shown here, the other caspase 3 blots are reported in Supplementary data, (Figure 2). (d) Western blot of cleaved caspase 3 was assayed at 4, 8, and 12 h (25 ng/ml FasL) using anti-caspase 3 polyclonal antibody (no. 9662). N=2