Cul4b

5637 BC cells were divided into control, siRNA-NC (non-specific siRNA with scramble), si-CUL4B (silencing CUL4B), CUL4B (CUL4B overexpression vectors, PLOC-CUL4B purchased from Invitrogen, CA, USA), WIF-1 (100ng/ml, an inhibitor of the Wnt/β-catenin signaling pathway, Sigma-Aldrich Chemical Company, St Louis, Missouri, USA) and CUL4B + WIF-1 groups (transfected with CUL4B and WIF-1) depending on the vector used for transfection. The siRNA sequences were synthesized using GenePharma (Shanghai, China) as follows: siRNA-NC: 5’-UUCUCCGAACGUGUCACGUTT-3’, siRNA-CUL4B: 5’-CCACCCAGAAGUCAUUAAUTT-3’. The 5637 cells or 5637-EGFP cells showing a stable expression of EGFP were used for establishing nude mouse metastatic models of BC in the logarithmic growth phase, which were plated and then transferred into an antibiotic-free medium 24 h later. The medium was replaced with serum-free Opti-MEM once cell density reached a rate of 40-60%. The transfection was conducted after mixing transfection reagents and plasmids or vectors (200 μl) at a cell density of 70% according to the instructions of Lipofectamine 2000 (11668-027, Invitrogen Inc., Carlsbad, California, USA). The samples were cultured at 37°C with 5% CO₂ for 6 h and upon completion, the medium was replaced with the normal medium containing serum.

ChIPs were performed as described previously^{19 (link)}. The Antibodies are CUL4B (Sigma, C9995), DDB1 (Santa, sc-137132), H3K27me3 (CST, 9733S), H3K4me3 (CST, 9751S), EZH2 (CST, 5246S), H2AK119ub1 (CST, 8240S), and H3 (Abcam, ab1791). The primer sequences are listed in Supplementary Table S4.

Total proteins were extracted from the cells using RIPA buffer (Keygen) with 1 mM proteinase inhibitor PMSF (Keygen) and cocktail (Roche). Forty micrograms of protein were separated on a polyacrylamide gel and transferred to a nitrocellulose membrane. The membranes were blocked for 1 h at room temperature in TBST containing 5% BSA, and then incubated overnight at 4 °C in TBST containing 5% BSA and following antibodies: CUL4B (Sigma, C9995); CD44 (Sigma, HPA005785 or Abcam, ab157107); NOTCH1 (CST, 3608S); NUMB (Abcam, ab4147); MYCN (Genetex, GTX133721); and GAPDH (CST, 5174S). Membranes were washed in TBST, incubated with a secondary antibody and conjugated with horseradish peroxidase for 1 h at room temperature. After washes with TBST, bands were detected using a SuperSignal Chemiluminescence kit (Thermo). Intensity of bands was qualified with GAPDH as the reference by Quantity One software.