FIG. 3.

Serum-starved HeLa cells also secrete a negative regulator of the PI3K-Akt pathway. (A) Serum-free medium, IGF-1, or CM from HeLa or HeLa-PH cells was untreated or heat treated, and their abilities to activate Akt were tested. (B) The CM from HeLa-PH or its column fraction was added to HeLa cells grown in serum-rich medium (20% volume of the medium) and incubated for 30 min prior to analysis for phospho-Akt. For the Centriprep column retentate (RT), the CM was concentrated with the Centriprep 10K or 50K filter column. Each concentrate (10×) was added directly to HeLa cells grown in serum-rich medium. (C) The HeLa cell CM collected after 48 h or 72 h of serum starvation was untreated or heat treated, and its ability to activate Akt was assayed.