Cell Culture and Reagents
The human TCam-2 cell line was derived in 1992 from a testicular tumor sample of a pure classical seminoma [21]. TCam-2 (obtained from Prof. Dr. Hubert Schorle, Department of Developmental Pathology and Department of Molecular Diagnostics Institute of Pathology, Bonn Medical School, Germany) was grown in RPMI 1640 medium (Gibco, #11875093) supplemented with 10% FBS (fetal bovine serum) (Gibco, #16000044), 1% penicillin/streptomycin (Gibco, #15140122) and 200 mM glutamine (Gibco, #25030081). Cells were grown at 37°C and under 5% CO2 humidified atmosphere. Rapamycin (Glentham Life Sciences, #GA7417) was dissolved in DMSO (Sigma, #D8418) at a stock concentration of 10 mM. Experimental groups were: control (no rapamycin treatment), 4 nM, 20 nM, 100 nM, 500 nM and 1000 nM rapamycin for 48 h and 72 h [22].
Western blotting
Western blot was performed as described previously [23]. In summary, a total of 4 × 105 cells/well were seeded on six-well plates and let attached overnight. Total protein from the TCam-2 cells were extracted using RIPA lysis buffer (SantaCruz, #sc-24948) supplemented with protease (Halt Phosphatase Inhibitor Cocktail, Thermo Scientific, #78446) and phosphatase inhibitor cocktails (Halt Phosphatase Inhibitor Cocktail, Thermo Scientific, #78420). After the total protein concentration was determined, 20 μg of total lysate was loaded into each lane, separated electrophoretically using NuPAGE 4-12% Bis-Tris gel (Invitrogen™, #NP0321BOX), and then electroblotted onto PVDF membrane (Thermo Scientific, #88585). Later, membrane was blocked in TBS-T buffer (0.1% Tween-20 in Tris-buffered saline) with 5% nonfat dry milk (Sigma, #70166) for one hour. The membrane was then incubated with appropriate primary antibodies anti-mTOR (1:1000 dilution; Cell Signaling, #2983), anti-p-mTOR (1:1000 dilution; Cell Signaling, #2971), anti-P70S6K (1:1000 dilution; Cell Signaling, #9202), anti-p-P70S6K (1:1000 dilution; Cell Signaling, #701064), anti-PCNA (1:1000 dilution; Cell Signaling, #2586) and anti-Caspase-3 (1:1000 dilution; Cell Signaling, #9662) overnight at 4°C. After incubation with primary antibody, membrane was washed with TBS-T for three times for 10 min each, incubated with HRP-conjugated secondary antibody (1:5000 dilution, Cell Signaling, #7074) for one hour at room temperature and subsequently washed with TBS-T. Phospho-specific antibodies were always used for the first round of probing. Membrane was strippped with western blot stripping buffer (Takara Bio, #T7135A). Immunobolt images were acquired and bands were quantified by using Image Lab Software (BioRad, #1708265). All experiments were repeated at least three times. β-Actin was used as an internal control and ratio of expression level of individual protein to that of β-Actin from the same samples was used to determine the final expression levels each protein.
Wound-Healing Scratch Assay
A total of 5 × 105 cells/well were seeded on six-well plates and let attached overnight. After overnight attachment, a straight line was created by scratching with a 100 μL pipette tip. Then the medium was removed, fresh medium with respective drug concentrations were added and images were obtained for t=0 h. The migration/healing was determined for 48 and 72 hours by obtaining microscopy images of the same area (Axiovert 135; Carl Zeiss Microscopy). Migration/healing was determined as previously described by using Fiji [24] and the percent healing against the control group was determined.
Assesment of Apoptosis
Viability, apoptosis and necrosis were evaluated by Annexin V/Propidium Iodide (PI) staining. For this purpose, cells were seeded into six-well plates as 5 × 105 cell per well and incubated for overnight for attachment. Cells were then treated with rapamycin at respective doses for 48 and 72 hours. For staining, cells were detached by trypsinization, washed once with Dulbecco’s phosphate buffered saline solution (DPBS, Thermo Fisher Scientific, #14190144) and suspended in 1X ice cold Annexin V Binding Buffer (BioVision Inc., #1006) followed by labelling with 5 μL Annexin V-FITC reagent (Biolegend, #640906) and 1 µL PI solution (Thermo Fisher Scientific, #P3566, diluted to 250 μg/mL in DPBS) by incubating for 10 minutes under dark conditions at room temperature. Cells were read with DxFLEX flow cytometry system (Beckman Coulter Inc.) and analysed by CytExpert software [25]. The experiment was done as triplicates and 2.5 × 104 cells per analysis were acquired at medium flow rate.
Cell cycle Assessment
DNA content analysis was performed by using Cell Cycle Kit (Beckman Coulter Inc., #C03551). Cells were seeded as 5 × 105 cell/six-well plate and incubated overnight for attachment. After incubating with rapamycin at respective doses for 48 and 72 hours, cells were detached by tyripsinization, washed once with DPBS, fixed with ice cold 70% ethanol by adding dropwise followed by incubation at 4°C for an hour. Tubes were stored at -20 °C overnight, and ethanol was removed by centrifuging cells at 400 x g for 5 minutes. Pellet was suspended in 500 µL cell cycle kit reagent, and tubes were incubated at room temperature under dark for 30 minutes. DNA content was analyzed by DxFLEX flow cytometry system (Beckman Coulter Inc.) and analysed with ModFit LT 5.0 software (Verify Software House) [26]. The experiment was done as triplicates and 5 × 104 cells per test were acquired at medium flow rate. Analyses were performed
Statistical Analysis
The statistical analysis was performed using GraphPad Prism 7.0 (GraphPad Software). Data were analyzed by using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison tests. A p value lower than 0.05 was considered statistically significant.