LBY135 was supplied from Novartis (Basel, selleck chem Switzerland), Sorafenib (BAY 43-9006) from Bayer (Leverkusen, Germany). Viability test Cell viability was determined by a colorimetric 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. HCC cell lines were seeded onto 96-well plates. On day one after seeding, cells were treated as indicated. We added 10 ��L MTT (5 mg/mL) 48 h after treatment and incubated cells for a further 3 h at 37��C. Next, supernatant was discarded and cells were lysed by adding 100 ��L 1-propanol to each well followed by shaking plates till complete lysis. Absorbance was measured at 550 nm in a microtiter plate reader. A viability of 1 was defined as the absorbance of untreated cells.

Coating of microtiter plates To ease ligand-receptor interaction with the crosslinking supplement IgG F(ab)��2, 96-well plates were coated with IgG F(ab)��2 before seeding cells. One hundred microliters of sterile filtered 100 nmol/L sodium bicarbonate buffer (pH 9.2) containing 5 ��g/mL IgG F(ab)��2 was added to each well and incubated for 2 h at room temperature (RT). After replacement of F(ab)��2 buffer by cell culture media, plates were stored at 4��C. Coated plates were stable for at least 1 wk. RNAi and transfection To knock-down protein expression, we administered specific small interfering RNA (siRNA) against MCL-1 or BCL-xL. As a control we used siRNA specific for green fluorescent protein (GFP).

The following siRNA sequences were applied (MWG Biotech, Ebersberg, Germany): BCL-xL, 5′-gcuuggauaaagaugcaaTT-3′ (sense) and 5′-uugcaucuuuaucccaagcAG-3′ (antisense), MCL-1, 5′-aaguaucacagacguucucTT-3′ (sense) and 5′-gagaacgucugugauacuuTT-3′ (antisense), GFP, 5′-ggcuacguccaggagcgcaccTT-3′ (sense) and 5′-ggugcgcuccuggacguagccTT-3′ (antisense). Here, capitals represent deoxyribonucleotides and lower case letters represent ribonucleotides. Huh7 cells were seeded onto 12-well plates and after 24 h transiently transfected in OPTIMEM with Lipofectamine RNAiMax (Invitrogen) according to the manufacturer��s protocol. Expression levels were analyzed 24, 48 and 72 h after transfection via Western blotting analysis. Detection of apoptosis HCC cells were seeded onto 12-well plates and treated as indicated 1 d after transfection. Forty eight hours after treatment, cells were washed with cold PBS, collected and resuspended in a hypotonic buffer containing 0.1% (w/v) sodium citrate, 0.1% (v/v) Triton X-100, and 50 ��g/mL Propidium Dacomitinib iodide (PI, Sigma). After 3 h incubation at 4��C, nuclei from apoptotic cells were quantified by fluorometric absorbance cell sorting according to the protocol of Nicoletti et al[24]. Cell lysis and Western blotting Cell lysis, SDS-PAGE and Western blotting were performed as described previously[13].