Ac h3 lys9

For immunohistochemical staining, the slides were incubated overnight with anti-acetyl histone H3 (Cell Signaling, Danvers, MA, USA) and then anti-rabbit secondary antibody for 60 minutes at RT (Vector laboratories, Burlingame, CA, USA). HRP was detected using the Vector DAB detection system, and the slides were counterstained with Mayer's hematoxylin. For immunofluorescence assays, the samples were fixed with methanol at −20°C for 6 minutes, blocked with 0.5% (v/v) Triton X-100 in PBS and 3% (w/v) bovine serum albumin (BSA) and incubated with anti-Vimentin (Thermo Scientific, Waltham, MA, USA), anti-BMI-1 (Millipore, Billerica, MA, USA), anti-Pan-cytokeratin (Cell Signaling, Danvers, MA, USA), anti-phospho S6 (Cell Signaling, Danvers, MA, USA) and ac.H3 (Lys9) (Cell Signaling, Danvers, MA, USA). Cells were then incubated with FITC- or TRITC-conjugated secondary antibody and stained with Hoechst 33342 (Sigma-Aldrich Corp., St. Louis, MO, USA) to visualize DNA content. Images were taken using a QImaging ExiAcqua monochrome digital camera attached to a Nikon Eclipse 80i Microscope (Nikon, Melville, NY) and visualized with QCapturePro software.