[HELP with High Resolution Image Viewing]

[View Larger Version of this Image]

Figure S3. Tm treatment inhibits budding when added immediately after α-factor release but does not affect budding when added 30 min after α-factor release. Wild-type (MNY 1005) cells were synchronized in G1 by α-factor treatment and treated with Tm either immediately after α-factor release (+Tm at 0′) or 30 min after α-factor release (+Tm at 30′). (A) Schematic representation of two conditions of synchronized Tm treatment. (B) Percentage of total cells that contained a bud during all three treatments. Note that cells treated with Tm immediately after α-factor release were budding delayed (no substantial budding until 60 min after α-factor release) compared with untreated cells. Tm treatment 30 min after α-factor release did not affect budding. (C) Flow cytometric analysis of cells stained with Sytox green to measure DNA content for the first hour after α-factor release for all three treatments. Note that under both conditions of Tm treatment, cells replicated their DNA at the same rate as untreated cells. Therefore, the delayed budding in cells treated immediately with Tm represents a direct block in bud formation rather than a delay in S-phase entry. All error bars represent the SD of three repeats. Because the addition of Tm at 30 min after α-factor release did not affect the budding process, we used this condition in subsequent experiments to characterize the effects of Tm treatment on cell cycle events after bud formation.