OCL formation in cocultures of normal OCL precursors with p62^−/− stromal cells treated with TNF-α. (A) CFU-GM cells (10⁴ cells/well) from spleens of p62^+/− mice were cultured with stromal cells from p62^+/− and p62^−/− mice for 7 days in the presence of TNF-α. The cells were then stained for TRAP, a marker enzyme of OCLs, and the number of TRAP⁺ multinucleated cells determined. Results represent the mean (± SEM) for 4 determinations. *P < .01 compared with cells cocultured with p62^+/− cultures. (B) RANKL production by p62^+/− and p62^−/− mouse stromal cells. Stromal cells from long-term mouse marrow cultures were treated with media alone or TNF-α. Conditioned media were harvested 4 days after the start of the cultures. The concentration of soluble RANKL present was determined using an ELISA kit for mouse RANKL. Results are reported as RANKL concentration (pg/mL) and are the mean (± SEM) of 4 samples. *P < .01, compared with p62^+/− stromal cell cultures. Similar results were seen in 3 independent experiments.