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mTOR Controls Ovarian Follicle Growth by Regulating Granulosa Cell Proliferation

Figure 2

mTOR inhibition by RAP in Rat SIGC.

Treatment with RAP is shown to inhibit mTOR activity by reducing phosphorylation of the target residue of p70S6 kinase (A) and 4E-BP (B). As low as 0.1 nM RAP significantly (P0.05, one-way ANOVA, letters denote bars that significantly differ from one another) reduces P-p70S6K and P-4E-BP eighteen hours after treatment. Cells were similarly treated with a dose curve of RAP (C–E) and were counted (C) or processed for biochemical viability assay (D) at 18 and 48 hours post-treatment. High doses (100 and 1000 nM) of the drug resulted in significantly (P0.0001, one way ANOVA with Bonferroni's Multiple Comparison Test, denoted a, b) reduced cell number and viability at 48 hours compared to all other treatments (denoted by a,b). Low doses (1E-3 and 1E-5 nM) of RAP resulted in non-significant but consistently increased cell number at 18 and 48 hours. Washout of RAP after 3 hours of treatment resulted in reduced, but still significant reductions in cell viability 48 hours post-treatment (E). Altered SIGC proliferation correlated with altered cell cycle parameters (F). Cells were grown in the dose curve of RAP. 18 hours post-treatment, cells were fixed and processed for DNA content cell cycle analysis via flow cytometry. Average results from three individual experiments are shown in F. As seen in other cell types, a dose-responsive induction of cells in the G1 stage of the cell cycle occurred with RAP treatment. Significantly different percentages of cells in G1 were found (P0.05, one way ANOVA with Bonferroni's Multiple Comparison Test, significantly different populations denoted by a,b,c) with increasing RAP. (G) RAP does not induce Caspase activity in Rat SIGC. SIGC were treated with either vehicle (VEH) or specified concentrations of RAP or Camptothecin (CT - positive control for apoptosis induction). Cells were collected at 24 and 48 hours post-treatment and processed for plate based chemiluminescent Caspase activity measurements. Relative Luminescence units are graphed for each sample per time point. Data shown are the average of two unique experiments.

doi: https://doi.org/10.1371/journal.pone.0021415.g002