Fig 2: Effect of Si-Sema6c on the PI3K-AKT-rpS6 signal pathway. (A-C), the morphology of ovaries treated by NC-SiRNA, Si-Sema6c or Si-Sema6c with LY294002 after 4 days of culture. (D,E) The numbers of primordial follicles and growing follicles were determined according to HE staining and MVH/ZP3 labelling. In the Si-Sema6c group, the number of primordial follicles decreased and the number of growing follicles significantly increased (*P < 0.05). But when we added LY294002, the number of primordial follicles increased and the number of growing follicles significantly decreased compared with the Si-Sema6c group (*P < 0.05). The Si-Sema6c-LY294002 group showed similar follicle number with the Con group (P > 0.05). The two methods (HE staining versus MVH/ZP3 labelling) showed no significant difference (P > 0.05). (F) The levels of AKT, p-AKT (S473), rpS6, p-rpS6 (S235/236) were examined by Western blot analyses. (G) Quantification of Western blotting indicated an enhanced phosphorylation of AKT at Ser473 and rpS6 at Ser235/236 in Si-Sema6c group compared with the Con group (**P < 0.01). LY294002 reversed the increased phosphorylation of AKT and rpS6 caused by the Si-Sema6c (**P < 0.01). The data presented at panels (D, E, G) correspond to mean ± S.E.M. from three independent experiments.

Fig 3: RPH1 is genetically involved in the TORC1 pathway. (A) The indicated strains were spotted on YPD plates with low dose of rapamycin. Pictures were taken after grown 2 or 4 days. (B) Tet-off system derived yeast strains were spotted on plates with the indicated doses of rapamycin in the presence of doxycycline. (C) Log-phase cells were cultured under rapamycin treatment, and were isolated at the indicated times and subjected to immunoblot with different antibodies. G6PDH served as a loading control. Asterisk indicates a non-specific band. (D) The phosphorylated Rps6 levels relative to total Rps6 levels in panel C were plotted with the indicated time points and then normalized to the time 0 point of individual strain by densitometric analysis using ImageJ software. The error bars represent mean ± SD from three independent biological repeats. t-test, **P < 0.01.