P pten

The CMT-U27 cells were cultured on coverslips coated with 1% gelatin. Cells were fixed with 2% paraformaldehyde for 20 min at 4ºC and permeabilized with 0.5% Triton X-100 in PBS for 10 min. The cells were blocked with 5% animal serum (donkey or goat) in 2% bovine serum albumin in PBS for 1 h at room temperature. Subsequently, the cells were incubated with primary antibodies against anti-p-PI3K (Affinity Biosciences) and p-PTEN (Santa Cruz Biotechnology) in blocking solution at 4ºC overnight. Then, the cells were incubated with Dylight® 488 conjugated goat anti-rabbit IgG (Bethyl Laboratories) and Cy3-conjugated donkey anti-mouse IgG (Jackson ImmunoReasearch). Nuclei were stained with 4',6-Diamidino-2-Phenylindole (DAPI). The cells were then mounted with mounting medium (Dako, Carpinteria, CA, USA), and images were captured using a confocal microscope (Carl Zeiss, Inc., Jena, Germany). We used the software ImageJ (version 1.52a) to measure the mean fluorescence intensity of each channel in three different regions. The relative fluorescence intensity was represented as compared to the control.

Antibodies against p-PTEN (Ser 380/Thr 382/383), PTEN, p-Akt, Akt, proliferating cell nuclear antigen (PCNA), cyclins D1, A, B and E, beta-actin, tubulin, follicle stimulating hormone (FSH) receptor, p53, early growth response protein 1 (Egr-1) and peroxisome proliferator-activated receptor gamma (PPARγ) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). A TUNEL assay kit was purchased from R&D Systems.
FSH (working in 10 ng/ml), insulin (working in 500 nM), Dehydroepiandrosterone (DHEA) solution (1.0 mg/mL in methanol ), 5α-Dihydrotestosterone (DHT) solution (1.0 mg/mL in methanol), and Bicalutamide (working in 50 μM) were all purchased from Sigma (Sigma, St Louis, MO).

Mouse anti-caspase-9, Bcl2, Bax, AKT, p-AKT and anti-PARP antibodies were from Cell Signaling Technology. mTOR, p- mTOR, PTEN, p-PTEN, p38, p-p38, CFLAR were from SANTA CRUZ. DR4 and DR5 were from Boster. Rabbit anti ISG12a antibody was from Abcam (ab171919). Mouse anti-β-actin and anti-V5 monoclonal antibody were from Sigma and Invitrogen respectively. Pancaspase inhibitor z-VAD-FMK was obtained from Promega. 2'-O-me-anti-miR-942 (5'-CACAUGGCCAAAACAGAGAAGA-3') & 2'-O-me-GFP miR (5'-AAGGCAAGCUGACCCUGAAGU-3') were from Takara. The Annexin V-FITC apoptosis detection kit and TRAIL were purchased from BD Pharmingen and R&D Systems respectively.