Overexpression of p27KIP1 as well as disruptive GM-CSF/PI3K/mTOR signaling down-regulate mcl-1 protein expression.
(A) DCs were left untreated or infected with rAd-LacZ or rAd-p27. After 48 hours of culture in the presence of IL-4 and GM-CSF, DCs were harvested for the preparation of whole cell lysates. Then equal amounts of protein (25 μg/lane) were loaded, and the levels of mcl-1 were determined. Data shown are representative for 2 independent experiments with different donors. (B) DCs were cultured either in IL-4 (No GM-CSF) alone or IL-4 and GM-CSF. Cultures containing IL-4 and GM-CSF were supplemented with or without 10 μM LY294002, 1 μM Rapa, or DMSO. Equal amounts of protein (20 μg whole cell lysate/lane) were loaded, and the levels of mcl-1 were determined. Data shown are representative for 3 independent experiments performed with different donors. (C) Quantification of mcl-1 expression in control and Rapa-treated DCs after either 24 hours or 48 hours of culture as described in “Materials and methods.” Data shown are representative for 4 independent experiments performed with different donors.
