FIG 1.

Rapamycin induces rapid LD replenishment. Wild-type (BY4741) yeast cells were pregrown for 48 h to stationary phase and then inoculated into fresh YPD medium at low density (OD₆₀₀ of 0.25). Rapamycin (100 ng/ml) (•) or vehicle alone (○) was added after 6 h of growth (A) or at the time of seeding (B). The LD indexes were measured using the LFR assay and normalized to that obtained in pregrown stationary-phase cells (relative LD index, or rLD) (upper). Growth was determined by absorbance at 600 nm (Abs_600nm) (lower). (C) Aliquots from the control culture or rapamycin-treated cells after 2 h of rapamycin treatment, from the experiment depicted in panel A, were analyzed by confocal fluorescence microscopy. Cells were stained with BODIPY (green) and 4′,6-diamidino-2-phenylindole (DAPI) (blue) (scale bar, 2 μm). (D) Intensity of fluorescence was determined as described in Materials and Methods. Data are from three independent experiments ± standard deviations (*, P < 0.05; **, P < 0.01; ***, P < 0.001 for the control versus rapamycin treatment).