,22 and siRNA against GFP was designed from RiboBio Co., Ltd. (Guangzhou, China). The inhibitor of Myc/Max dimerization 10058-F4 (sc-213577) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). For transfection, plasmids (1-5 μg), siRNAs (100 nM final concentration), miRNA mimics (50 nM final concentration), or miRNA inhibitors (100 nM final concentration) were transfected into appropriate cells using Lipofectamine

2000 (Invitrogen), 48 hours after transfection cell pellets were collected Palbociclib nmr and subjected to RNA isolation and immunoblot analysis. RNA was extracted using TRIzol (Invitrogen) from the indicated cell lines according to the manufacturer’s protocol. DNA contamination was removed with RNAse-free DNase I. All reagents

for stem-loop RT were obtained from Promega, Inc. (Madison, WI) and RiboBio. PCR products were analyzed on 3% agarose gels. Small RNA U6 was used as an internal control. Real-time PCR was performed with SYBRGreen (Bio-Rad). Primers and other reagents of mature miRNA assays were purchased from RiboBio. Primers of real-time PCR for other genes are listed in Supporting Table 1. Chromatin immunoprecipitation (IP) assays were performed according to Yi et al.23 Briefly, intracellular protein-DNA complexes were cross-linked in situ by the addition of 1% of formaldehyde. Total lysates were then sonicated and subjected to chromatin-conjugated IP using specific antibodies. After reversal of cross-links, Selleckchem AZD1152 HQPA precipitated DNA was purified and analyzed by real-time PCR with specific primers (Supporting Table 1.). Cell cycle analyses were performed on propidium iodide–stained selleck inhibitor nuclei using a MoFlo XDP-Flow Cytometer (Beckman Coulter, Inc). Data were analyzed by single-histogram statistics.20, 24 For colony assays, 1 × 103 cells of the indicated type were plated in triplicate in soft agar (0.35% low melting point agarose on top of 0.7% bottom agarose) in six-well plates and fed intermittently with DMEM. Colonies were

enumerate after 2 weeks by staining with methylene blue after methanol fixation.25 Four-week-old male BALB/c nude mice were purchased from Shanghai SLAC Laboratory Animal Co. and maintained in microisolator cages. Tumorigenicity assays and tumor volume measurements were performed as previously described.20 Briefly a total of 1 × 107 indicated cells were suspended in 100 μL serum-free DMEM and injected subcutaneously in the flanks of animals. Tumor growth was monitored every three days for a total period of 30 to 40 days. Tumor volumes were calculated by the equation V (mm3) = a × b × c/2, where a is the length, b is the width, and c is the height. Informed consent was obtained at the Union Hospital in Wuhan and at the Eastern Hepatobiliary Surgery Hospital in Shanghai, China. The diagnosis of HCC was confirmed in each case by histological reviews. None of the patients received chemotherapy prior to hepatectomy. Co-IP was performed as described.