15 Whole-cell extracts from cultured cells or tissues were prepared and subjected to western blot. For immunodetection, the following antibodies were used: anti–Bcl-xL antibody and anti-human Mcl-1 antibody from Santa Cruz Biotechnology (Santa Cruz, CA); anti-mouse Mcl-1 antibody from Rockland (Gilbertsville, PA); anti-Bid antibody, anti-Bax antibody, and anti-cleaved caspase-3 antibody from Cell Signaling Technology (Beverly, MA); anti-Bak antibody from Millipore (Billerica, MA); anti-Bim antibody from Assay

Design (Ann Arbor, MI); anti-ubiquitin-specific peptidase 9, X-linked (USP9X) antibody from Abnova (Taipei, Taiwan); and anti–beta actin RAD001 antibody from Sigma-Aldrich (St. Louis, MO) or Cell Signaling Technology. To produce a xenograft tumor, 3 × 106 to 5 × 106 Hela–Bcl-xLTet-on clone A or Huh7 cells were subcutaneously injected to Balb/c nude mice. For induction of HA–Bcl-xL, the mice that

were injected with Hela–Bcl-xLTet-on clone A cells were fed with water containing 100 μg/mL doxycycline. For anticancer therapy, ABT-737 was administered as described.17 Sorafenib tablets were crushed and orally administered with water containing 12.5% Cremophor EL (Sigma-Aldrich) and 12.5% ethanol. We estimated the volume of the xenograft tumor using the following formula: tumor volume = π/6 × (major axis) × (minor axis)2. Hepatoma cell lines were transfected with Stealth select RNAi (set of three oligonucleotides, Invitrogen) Selleckchem Olaparib RNA interference (RNAi) directed against Mcl-1 or USP9X. A Stealth RNAi negative control kit (set of three oligonucleotides, Invitrogen) was used as a control for sequence-independent 上海皓元 effects following Stealth RNAi delivery. The transfections were carried out using Lipofectamine RNAiMAX (Invitrogen) according to the reverse transfection protocol. Real-time reverse-transcription

PCR (RT-PCR) was performed as previously described.15 Mcl-1 messenger RNA (mRNA) expressions were measured using TaqMan Gene Expression Assays (Assay ID: Hs03043899_m1) and were corrected with the quantified expression level of beta actin mRNA measured using TaqMan Gene Expression Assays (Assay ID: Hs99999903_m1). Data are presented as mean ± standard deviation. Differences between two groups were determined using the Student t test for unpaired observations unless otherwise noted. Multiple comparisons were performed by analysis of variance followed by Scheffe post hoc correction. P < 0.05 was considered statistically significant. Research has shown that Bcl-xL overexpression confers resistance to apoptosis in a variety of tumor cells. To examine its impact on tumor growth in vivo, we generated the Hela–Bcl-xLTet-on cell line which expresses the modified tetracycline repressor molecule (rtTA) and Bcl-xL under control of tetracycline-responsive cis-elements.