H 102

ChIP was performed as previously described, except extra crosslinking with 1.5 mM EGS for 30 min before addition of 1% formaldehyde [36 (link)]. Chromatin was incubated with anti-ZEB1 (5 μg, Santa Cruz H102, sc-25388X) and normal rabbit IgG control (5 μg, Santa Cruz, sc-2345) antibodies overnight and complexes were precipitated by protein A/G Dynabeads^® (Invitrogen 10002D/10004D, 25 μl each per IP). Precipitates were eluted (0.1 M NaHCO₃, 1% SDS) and chromatin was decrosslinked by first incubating 1 h at 37°C with 250 μg/ml RNaseA and 500 μg/ml proteinase K, followed by overnight incubation at 65°C. After DNA purification the indicated regions of NOG, FST and CHRDL1 promoters were amplified by quantitative PCR. HPRT1 was used as negative control [13 (link), 36 (link)].

IF was performed as previously described [70 (link)]. Briefly, MDA-MB-231 cells were seeded on glass coverslips in 35-mm Petri dishes and cultured until 50% confluence. They were treated with D-Fraction (IC₅₀, 12 h) or vehicle. After that, the cells were incubated with the primary antibody rabbit polyclonal anti-β-catenin (H-102) (Santa Cruz Biotechnology, sc-7199) for 1 h and then incubated with Alexa Fluor^® 568 goat anti-rabbit IgG (Molecular Probes, Invitrogen) for 1 h in the dark. Finally they were stained with 4’,6-diamidino-2-phenylindole (DAPI; 1:10000). Fluorescence images were acquired with a Nikon Eclipse E-600 microscope (Nikon, Melville, NY, USA), equipped with a SBIG model ST-7 camera (Santa Barbara Instrument Group). For each replicate, at least 400 cells in 400× random fields were evaluated and the proportion of cells expressing β-catenin in membrane or cytoplasm/nucleus was determined.

Cells grown in 8-well-chambered slide (Thermo Fisher) were fixed in 4% paraformaldehyde, then permeabilized with 0.2% Triton X-100. After washing and blocking, cells were stained with rabbit polyclonal anti-β-catenin antibody (1:1000, H-102, Santa Cruz, USA) and labeled with the secondary antibody conjugated with Alexa-Fluor-488. Counterstaining was performed with DAPI.