Ring1b

This study was approved by the Ethics and Research Committees of Shanghai Jiaotong University School of Medicine and was conducted in accordance with the Declaration of Helsinki Principles. Tissue microarrays (TMAs) purchased from ShGnghGi Outdo Biotech Company (China) were well-documented with clinicopathological information, including patient age, sex, tumor size and location, pathological grade, perineural invasion, lymph node metastasis, and tumor staging (detailed in Table 1), as well as follow-up data. Ninety pancreatic cancer patients included in the TMAs underwent pancreatectomy between September 2004 and December 2008, and postoperative follow-up was finished by December 2011. Ten cases without complete follow-up data were excluded. The average follow-up time of the eighty patients was 25.3 months (median, 14 months; range, 1–81 months). Microarrays consisting of one core, each 1.5 mm in diameter, were prepared by dot-arraying tissues from resected human PDAC tumors. Immunohistochemistry (IHC) staining was performed with specific antibodies according to the previous descriptions[29 (link), 30 (link)]. EZH2 (Millipore, #15217-662); Ring1B (#5694), H2A (#2578), ubiquityl-Histone H2A (Lys119) (#8240), and Tri-Methyl-histone H3 (Lys27) (#9733) antibodies were purchased from Cell Signaling Technology Inc.

These protocols have been performed following standard techniques. Ring1B, Fak and p63 antibodies were purchased from Millipore (Billerica, MA, USA). pY861-Fak antibody was from Biosource International (Life Technologies, Paisley, UK). Anti-activated MAP kinase (diphosphorylated Erk 1/2) was from Sigma (St. Louis, MO, USA) and Erk 2 (C-14) antibody was purchased from Santa Cruz Biotechnology (Heidelberg, Germany). Lamin B1 antibody was from Abcam (Cambridge, MA, USA) and Vinculin and α-Tubulin antibodies from Sigma (St. Louis, MO, USA). Fluorescent secondary antibodies were obtained from Invitrogen (Life Technologies, Paisley, UK) and Jackson ImmunoResearch Laboratories (West Grove, PA, USA). Rhodamine-Phalloidin was from Sigma (St. Louis, MO, USA). For focal contact counting, the cells were labeled with anti-Vinculin antibody and the Vinculin-positive patches were manually counted.
Western blot quantification was performed using Image J densitometry software. The intensity of individual bands was normalized to Lamin B or α-Tubulin signal, as a measure of protein relative abundance in the different samples and referred to control conditions.

The affinity-purified rabbit polyclonal antibodies for Cbx4 (Cat# GTX109662) and Cbx2 (Cat# GTX117711) were purchased from GeneTex, (Irvine, CA). The affinity-purified rabbit polyclonal antibody for H2AK119ub1 (Cat# 4889) was purchased from Cell Signaling (Beverly, MA). The affinity-purified rabbit polyclonal antibodies for H2A (Cat# ab18255), BrdU (Cat# ab1893), and Rybp (Cat# ab5976) were purchased from Abcam Inc (Cambridge, MA). The purified mouse monoclonal antibodies for H3K27me3 (Cat# 05-1591), Ring1B (Cat# D193-3), and phosphorylated histone H3 (pHH3) (Cat#, MABE939) were purchased from Millipore Corp. (Billerica, MA). The affinity-purified rabbit polyclonal antibodies for actin (Cat#.sc-1615) and Ki67 (sc-7846) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Secondary antibodies for Cy2-conjugated donkey anti-rabbit (Cat# 711-225-152); Cy3-conjugated donkey anti-rabbit (Cat# 711-165-152) and donkey anti-mouse (Cat# 715-165-150); and peroxidase-conjugated goat anti-rabbit (Cat# 705-035-147) and donkey anti-goat (Cat# 705-035-147) were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). peroxidase-conjugated goat anti-rabbit secondary antibody (ready-to-use) (Cat# 50-235) for IHC and biotinylated antibody for BrdU (Cat# 93-3944) were obtained from Zymed Laboratories Inc. (San Francisco, CA).