Fig. 2.

Extended release of rapamycin. (a) These arrays are stained with Hoechst 33342 and a cy3-anti-pS6 antibody [a downstream effector of mTOR (cells affected by rapamycin show a lack of S6 phosphorylation)]. For each spot, the largest radius of effect was determined by measuring pS6 per unit of area. Regions with average pS6 levels below a threshold were considered affected. Each line in the graph represents the concentration of rapamycin in relation to the radius of effect for each storage condition. (b) The quantitation of pS6 intensity and the number of cells per unit of area at 50-μm intervals from the center of a spot loaded with 7.5 μM rapamycin. The level of pS6 increases with increasing distance from the center of the spot, suggesting that a concentration gradient has been established. Consistent with the known effects of rapamycin, the spots have a mild antiproliferative effect on cells in a dose-dependent manner.