Figure 5
Paracetamol plus TRAIL induce cell death in primary murine as well as human LSECs in a Bim- and caspase-dependent manner. Murine and human LSECs were pretreated (30 min) with Z–VAD–fmk and stimulated with 10 mM paracetamol, 30 ng/ml TRAIL or the combination thereof for 16 h, and visualized microscopically after Hoechst 33342 and PI staining. Inserts show a two-fold magnification from the center of the picture (a). Cells were treated with 10 mM paracetamol and 30 ng/ml TRAIL, or the combination of both for 6 h; and Bim, cleaved caspase 3 and Tubulin as loading control, were detected by western blot (b, e). Cells were stimulated for 6 h with 10 mM paracetamol and bim-mRNA expression was measured by quantitative RT-PCR. GAPDH was used to normalize bim-expression levels (c). Murine and human LSECs were stimulated as described and DEVD cleavage was measured after 6 h (d, f). Mean values±S.D. triplicates are shown for DEVDase assay. A typical experiment out of four is shown for microscopy analysis. Western blot for Bim was repeated two times, yielding similar results.Mean values±S.D. triplicates of independent pooled LSEC isolation are shown for RT-PCR
