4.2. Spheroid Assay, Plasmid Transfection, and Western Blot Analysis

For the spheroid assay, 1.0 × 10⁴ cells/well were cultured in a U-bottom low-adhesion 96-well plate (S-Bio, Hudson, NH, USA) for 24 h. We transfected Lrp5 plasmids (#115907, Addgene, Watertown, MA, USA), β-catenin plasmids (#31785, Addgene), and IL-1ra plasmids (RG218518, Origene, Rockville, MD, USA) to MLO-A5 osteocytes and Snail plasmids (#31697, Addgene) to EO771 mammary tumor cells. All plasmids were transfected to approximately 2 × 10⁶ cells using lipofectamine 3000 (Life Technologies), and pcDNA was used as a control plasmid. In western blot analysis, cells were lysed in a radio-immunoprecipitation assay buffer and isolated proteins were size-fractionated and electro-transferred. We used antibodies against Snail, TGFβ, Lrp5, Runx2, Caspase 3 (cas3), cleaved Caspase 3 (c-cas3), histone H4 (Cell Signaling, Danvers, MA, USA), p53, CXCL2, IL1β, IL1ra (Invitrogen, Carlsbad, CA, USA), MMP9 (Santa Cruz Biotechnology, Dallas, TX, USA), TRAIL (Novus Biologicals, Centennial, CO, USA), LIMA1 (Novus Biologicals), DSP (Proteintech, Rosemont, IL, USA), CXCL5 (Abcam, Cambridge, MA, USA), and β-actin (Sigma-Aldrich). The uncropped gel images are shown in the supplementary information. RNA interference was conducted to silence histone H4 (Hist4h4 Select, Life Technologies), together with a nonspecific negative control siRNA (Silencer Select #1, Life Technologies). Cells were transiently transfected with siRNA with lipofectamine (Life Technologies), and the medium was replaced by a regular culture medium after 24 h.