Animals
The mice used in this study were 6 weeks of age, female BalbC mice. They were obtained from the animal center of Yeditepe University Medical School Experimental Research Center (YUDETAM) where they were housed in a 23-25 °C temperature room and on a 12 h light/12 h dark (on 7 am, off 7 pm, respectively) light cycle, and water ad libitum. The animal protocols used in this study were approved by the Institute Animal Care and Use Committee of Yeditepe University (Protocol number#200).
Granulosa cell culture
The mice were euthanized by cervical dislocation. Ovaries were dissected from female mice 48 h after injection of 5 units pregnant mare's serum gonadotropin (PMSG, Sigma, USA). Large antral follicles were then punctured with a sterile 26-gauge needle to obtain granulosa cells. After removal of oocytes, granulosa cells were washed with RPMI (Invitrogen, USA) and seeded at a density of 1×106 cells with RPMI medium containing 10% (v/v) fetal bovine serum and penicillin/streptomycin for 72 h at 37oC in the presence of 5% CO2.
Small interfering RNA (siRNA) transfection
After 72 h of culture, the granulosa cell, culture medium was changed and siRNA transfection was carried out according to Dharmafect (Thermo Scientific, USA) transfection reagent protocol. The siRNAs were designed by and purchased from Thermo Scientific. Cells were treated with 0.5 ml of culture medium containing DharmaFECT transfection reagent (Thermo Scientific) containing 10 μM ds-siRNAs (Dharmacon, Chicago, IL) against: c-Abl–5’AGGUGAAAAGCUCCGGGUC3’. siCONTROL NON-TARGETING pool siRNA (Dharmacon) against cyclophilin B was used as the transfection control. We also used on target plus non-targeting siRNA as a negative control. siGLO green transfection indicator and Dharmafect transfection reagent were used for transfection. After 24 h of incubation, we detected c-Abl siRNA transfected cells by fluorescence imaging.
Immunofluorescence
Mouse ovaries were fixed with 4% paraformaldehyde for 6 hr, dehydrated, and embedded by paraffin. Paraffin-embedded mouse ovary samples were cut into 5 μm sections and incubated overnight at 56°C. Tissue sections were deparaffinized in xylene and rehydrated in a graded series of ethanol’s while antigen retrieval was performed by microwaving in EDTA (pH: 8.0). Antigen retrieval was performed in microwaving in EDTA (pH: 8.0) and Slides were then incubated in a humidified chamber with TBS-T (Tris-buffered saline containing 0.1% Tween-20 and 5% normal goat serum; Sigma, St Louis, MO) for 1 hour at room temperature. Anti-c-Abl and anti-mTERT antibodies were used at 1/250 dilution for overnight incubation at 4oC in 5% normal goat serum (NGS)/PBS. Control sections were incubated with normal rabbit IgG serum (Vector Laboratories, Burlingame, CA) at the same concentration. Following steps were performed at room temperature, with PBS washes between incubations. Primary antibody binding was detected using anti-rabbit Alexa Flour-488-conjugated secondary antibodies (Thermo Fisher Scientific) diluted 1:250 in 5% NGS in PBS with 0.01% Tween-20 for 1 h at room temperature and incubated in DAPI for 5min at room temperature before imaging. For negative control, sections were treated with appropriate mouse IgG. Images were captured following confocal microscopy.
For immunocytochemistry, granulosa cell culture medium was removed, and cells were fixed with 4% PFA for 20 minutes at room temperature. Afterwards, cells were incubated with 5% NGS for blocking. Anti-c-Abl and anti-mTERT antibodies were applied at 1:250 dilution for overnight incubation at 4oC in 5% normal goat serum (NGS)/PBS. Primary antibody binding was detected using anti-rabbit Alexa Flour-488-conjugated secondary antibodies (Invitrogen) diluted 1:250 in 5% NGS in PBS with 0.01% Tween-20 for 1 h at room temperature and incubated in DAPI for 5min at room temperature before imaging. Sections were treated with appropriate mouse IgG for negative control. Images were captured following confocal microscopy.
Western Blot
Total protein from each granulosa cell culture plate was extracted using T-PER tissue protein extraction reagent (Pierce, Rockford, IL, USA), supplemented with protease inhibitor cocktail (1 mM Na3VO4, 10 μg/ml leupeptin, 10 μg/ml aprotinin and 1 mM phenylmethylsulphonylfluoride; Calbiochem, San Diego, CA, USA). The protein concentrations of granulosa cells from each group was determined by Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, CA, USA). Western blot analysis was performed as previously described [50]. Shortly, 20-μg of protein was loaded into each lane. To reduce the non-specific binding, the membrane was blocked with 5% non-fat dry milk in TBS-T buffer (0.1% Tween-20 in Tris-buffered saline) for 1 h. The membrane was then incubated with rabbit polyclonal c-Abl antibody (1:1000 dilution; Thermo Scientific, USA) and then, incubated in peroxidase-labeled goat anti-rabbit IgG (Pierce; USA) and subsequently washed and chemiluminescence detecting reagents were used for detection of c-Abl protein expression. We repeated the same procedure for mTERT (1:500 dilution; Thermo Scientific, USA) and PCNA (Proliferating Cell Nuclear Antigen) (1:1000 dilution; Cell Signaling Technology, USA). We used β-actin (1:1000 dilution; Thermo Scientific, USA) for internal control. Each experiment repeated 3 times.
Immunoprecipitation
Mouse granulosa cells were freshly isolated and directly subjected to protein extraction by using RIPA lysis buffer system (Santa Cruz) supplemented with protease inhibitor cocktail (Santa Cruz), sodium orthovanadate (Santa Cruz), phenylmethylsulfonyl fluoride (Santa Cruz) and Halt™ Phosphatase Inhibitor Cocktail (Thermo Scientific) according to manufacturers’ instructions. Protein concentration from the total lysate was determined by using Pierce™ Coomassie Plus (Bradford) Assay Kit (Thermo Scientific). Then, 100 µg total lysate was subjected to immunoprecipitation (IP) by using Pierce™ Crosslink IP Kit according to manufacturer’s instructions. Briefly, anti-c-Abl (ab15130, Abcam; 10 µg) or anti-TERT (MA5-16034 (Clone: 2C4), Thermo Scientific, 1:100) antibodies were coupled and crosslinked with Protein A/G resin. The uncoupled resin was used as a negative control. Then, 100 µg total granulosa cell lysate was precleared and either with uncoupled resin or coupled resin overnight at 4°C. The flow-through (FT) from the IP reaction was collected to confirm the IP and the antigen was eluted. The elute (E) and FT fractions were then resolved on 4-12 % Bis-Tris gel (Thermo Scientific) and subjected to western blot. Membranes were incubated with either anti-c-Abl (ab15130, Abcam; 2 µg) or anti-TERT (ab191523, Abcam; 10 µg) overnight at 4°C, washed, and incubated for 1 h at room temperature. The blot images were acquired by using a ChemiDoc™ XRS+ System (Bio-Rad).
Statistical analysis
Groups were compared by Student T-tests and One-way ANOVA adjusting multiple comparisons with Tukey method. All experiments were performed in at least three replicates. Statistical calculations were performed using GraphPad Prism 7 program.