Fig 1: Dr5−/− and Trail−/− mice are resistant to biliary injury following SM intrabiliary instillation. (a) In situ hybridization using mRNA detection probes for Trail and Dr5 performed on liver tissue sections of saline- or BV6-treated mice at day 5. The white arrows point to cholangiocytes. The orange arrows point to surrounding infiltrating cells (inflammatory cells, fibroblasts); BD, bile duct. (b) Representative images of liver sections from Dr5/- (top) and Trail−/− (bottom) mice injected with a single dose of BV6 or saline in the biliary tree and killed after 5 days. H&E staining, panCK IHC, picrosirius red-stain (collagen), α-smooth muscle actin IHC and Mac2 staining are depicted. Photomicrographs taken at × 20 magnification. The arrows point to the bile ducts; pv, portal vein. (c) Serum ALT, alkaline phosphatase, total bile acids and total bilirubin. Dr5−/−: saline (n=5), BV6 (n=5); Trail−/−: saline (n=4), BV6 (n=7). *P<0.05. (d) Il6, Il8, Il1β, Tnfα, Mcp-1/Ccl2, Cd68 and Coll-1α1 gene expression analyzed by qPCR in total liver lysates of wild-type, Dr5−/− and Trail−/− mice. Expression normalized to 18S rRNA. Data are expressed as fold increase over control. Mean±S.E. are depicted. *P<0.05, **P<0.005; NS, not significant
Fig 2: Normal human cholangiocytes are moderately sensitive to SM-induced, ripoptosome-mediated apoptosis. (a) Apoptosis assessed morphologically after DAPI staining (top panels) and by caspase 3/7 activation (bottom panels) in human cholangiocyte cell lines H69 and NHC, and the human breast cancer cell line MDA-MB-231, incubated for 24 h with or without (cnt) the SMAC mimetic TL32711 (1 μM), in the presence or absence of neutralizing antibodies against TNFα (1 μg/ml) or FasL (1 μg/ml), or recombinant TRAIL-R2:Fc (1 μg/ml). (b) TNFα and TRAIL gene expression analyzed by qPCR in H69, NHC and MDA-MB-231 cells incubated in the presence of TL32711 for the indicated times. Expression normalized to GAPDH RNA. Data are expressed as fold increase over control. Mean±s.e. are depicted from three independent experiments in H69 and NHC (c) H69 cells were treated with TL32711 and subjected to caspase 8 immunoprecipitation at the indicated time points. Affinity-purified proteins and total cell lysates (input) were analyzed by immunoblot for caspase 8, FADD and RIP1. (d) NHC cells were incubated with or without the TL32711 for 24 h, in the presence or absence of the pan-caspase inhibitor Q-VD-OPh (QVD, 10 μM) or the RIP1 kinase inhibitor necrostatin 1 (nec-1, 20 μM). Apoptosis was assessed morphologically after DAPI staining (left panel) and by caspase 3/7 activation (right panel). (e) H69 and RIP1−/− H69 (clones 1 and 2) were incubated for 24 h in the presence or absence of TL32711. Apoptosis was assessed by DAPI staining (left panel) and by caspase 3/7 activation (right panel). In (a, d and e), mean±S.E. are depicted from three independent experiments performed in triplicate. *P<0.05, **P<0.005
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