Stat3 sirna

U87 and U251 human glioblastoma cell lines were cultured in DMEM (life technologies, USA) supplemented with 10% fetal bovine serum (Sigma-Aldrich), 100 units/ml penicillin-100 μg/ml streptomycin (Sigma-Aldrich). The culture was maintained at 37 °C in a humidified atmosphere containing 5% CO₂. Temozolomide (TMZ) and Ly294002 were obtained from Sigma-Aldrich. S3I-1757 was kindly provided by Dr. Said Sebti (Moffitt Cancer Center). PIK-75, TGX221, IC-87114 and AS-605240 were purchased from Cayman Chemical (USA). G418 sulfate (50 mg/ml) was obtained from Life Technologies (USA). Stat-3 siRNA was obtained from Sigma-Aldrich (USA). Rabbit anti-Human-Akt, Rabbit anti-Human phospho-Akt, Goat anti-Rabbit IgG-HRP, anti- β-actin IgG-HRP and Rabbit anti-Human cIAP2 were obtained from Santa Cruz Biotech (USA).

STAT3 siRNA, PDGFR siRNA, and negative control siRNA were purchased from Sigma (USA). The cells were plated at 50% confluence, transfected with 50 nM siRNA complexed with Lipofectamine 2000 (Invitrogen) in Opti-MEM overnight, and then incubated for various amounts of time.

To ensure that siRNAs and overexpression vectors would be transfected into the inner cells of fetal ovaries, 0.5 μL of the siRNAs or overexpression vectors were first injected into isolated E15.5 fetal ovaries using glass pipettes with a stereomicroscope. After the ovaries were full of liquid, electrotransfection was performed by applying three 5-ms-long quasi-square pulses at a pulse-field strength of up to 30 V/cm. Rac1siRNA and STAT3 siRNA were purchased from Sigma. GDF9 siRNA and BMP15 siRNA pools were chemically synthesized (GenePharma Corporation, China), with sequences listed in Table S1. Control siRNA contained a scrambled siRNA sequence that did not lead to specific degradation of any known mouse mRNA. Ovaries then were cultured for four days to test for transfection efficiency of mRNA and protein levels using real-time PCR and Western blotting, respectively, or for eight days for histological examination and follicle counting.