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. 2014 Dec 23;9(12):e115618. doi: 10.1371/journal.pone.0115618

Figure 3. Testosterone inhibited the apoptosis via the Akt-FoxO3a pathway in the COV434 granulosa cells.

Figure 3

COV434 cells were incubated in phenol red-free DMEM supplemented with 2% Charcoal Stripped Fetal Bovine Serum (steroid-free conditions; Free) for 12 hours. The COV434 cells were then treated with vehicle or 10 or 100 nM testosterone for 16 hours for the RT-PCR analysis and 24 hours for Western blotting in the presence or absence of flutamide (10 µM) or LY294002 (25 µM) for one hour. A: (a) The mRNA expression of BimEL was determined using RT-PCR, and the beta-actin mRNA expression was used as an internal loading standard. Relative densitometric units of BimEL to beta-actin are shown in panel (d). The cell lysates were analyzed according to Western blotting using antibodies to BimEL (b), PARP (c) or beta-actin. Relative densitometric units of BimEL to beta-actin, and cleaved-PARP to total PARP are shown in panel (e) and (f), respectively. B: The cell lysates were also analyzed according to Western blotting using antibodies to phosphorylated-Akt, Akt (g), phosphorylated-FoxO3a or FoxO3a (h). Relative densitometric units of phosphorylated-Akt to total Akt, and phosphorylated-FoxO3a to total FoxO3a are shown in panel (i) and (j), respectively. Values shown represent the mean ± s.e. from at least three separate experiments. Significant differences are indicated by asterisks. *, p<0.05, **, P<0.01.