Anti bmi 1

Cells were placed on glass coverslips in 12-well plates and fixed with absolute methanol at −20 °C for 5 min. Spheres were prepared as described above. Cells and spheres were blocked with 0.5% (v/v) Triton X-100 in PBS and 3% (w/v) bovine serum albumin (BSA) and incubated with anti-Acetyl-Histone H3 (Lys9) (Cell Signaling, Danvers, MA, USA), anti-BMI-1 (Millipore, Billerica, MA, USA), and anti-Ki-67 (Cell Signaling, Danvers, MA, USA) as indicated. Cells were then washed three times and incubated with Fluorescein isothiocyanate (FITC) or Tetramethylrhodamine (TRITC)-conjugated secondary antibody for 60 min at RT and stained with Hoechst 33342 for visualization of DNA content. Images were captured using a QImaging ExiAqua monochrome digital camera attached to a Nikon Eclipse 80i Microscope (Nikon, Melville, NY, USA) and visualized with QCapturePro software.

Western blots and immunoprecipitations were performed as described previously (12 (link)). Antibodies for western blot were anti-EBNA3A (Abcam, ab16126, 1:1000 dilution), anti-EBNA3B (clone 6C9, Allday lab, E. Kremmer (39 (link)), 1:10 dilution), anti-EBNA3C (clone A10, gift from M. Rowe, University of Birmingham, 1:10 dilution), anti-γ-tubulin (Sigma, T6557, 1:8000 dilution), anti-BMI1 (Millipore, 05–637, 1:1000 dilution), anti-SUZ12 (Santa Cruz Biotechnology, sc-46264, 1:1000 dilution), anti-CBX4 (Santa Cruz, sc-517216, 1:1000 dilution) and anti-MEL18 (Abcam, ab5267, 1:1000 dilution). Antibodies for immunoprecipitations were anti-BMI1 (Bethyl Laboratories, A301-694A, 2 μg) and DYKDDDDK Tag antibody (NEB, 2368, 2 μg). RNA extraction was performed using Qiagen’s RNeasy mini kit, according to the manufacturer’s instructions. cDNA was obtained using Invitrogen’s SuperScript III First Strand Synthesis Supermix. QPCR for cDNA and DNA from ChIP was performed using Platinum SYBR green QPCR Supermix uracil DNA glycosylase (UDG) kit (Invitrogen), as described previously (12 (link)). Primers for STK39 (8 (link)), AICDA (40 (link)) and COBLL1 (10 (link)) loci and expression have been described before. All the oligonucleotide primers used are listed in Supplementary Table S1.

Whole-cell lysates were prepared using radioimmunoprecipitation assay (RIPA) buffer, immunoblotted as described,³² (link)^,33 (link) and analyzed using the following primary antibodies: anti-FAK, anti-phospho-p27 (S10), anti-p27, anti-c-Src, anti-β-actin, anti-α-tubulin, and anti-GAPDH (glyceraldehyde 3-phosphate dehydrogenase; Santa Cruz Biotechnology, Santa Cruz, CA, USA); anti-phospho-c-Src (Y416), anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-STAT3 (Y705), and anti-STAT3 (Cell Signaling, Danvers, MA, USA); anti-phospho-FAK (Y397) (Abcam, Cambridge, UK); anti-BMI1 (Millipore, Temecula, CA); anti-vimentin (Sigma, St. Louis, MO); anti-HA (Roche, Mannheim, Germany); and rabbit anti-human TM4SF5⁷ (link) and rabbit anti-mouse TM4SF5, which was produced using a peptide-corresponding mouse TM4SF5 (amino acid residues 117–138; CLIDNKWDYHFQETEGAYLRND) by ProSci (Poway, CA, USA).