Antibodies and reagents
Anti-microtubule associated protein 1 light chain 3 (LC3) (NB100-2220) and anti-Beclin 1 (ab55878) antibodies were purchased from Novus Biological (LLC, USA). Anti-Bcl-2-associated X protein (Bax) and anti-Bcl-2 antibodies were purchased from Abcam (Cambridge, MA, USA). Antibodies recognizing phospho-mTOR (Ser2448), mTOR, phospho-4E-BP1 (Thr37/46), 4E-BP1, phospho-p85S6K (Thr412), p85S6K, phospho-p70S6K (Thr389), and p70S6K were purchased from Cell Signaling Technology (Beverly, MA, USA). Lipofectamine 2000 (11668-019), LysoTracker Red (L7528) and MitoTracker (M7512) were purchased from Invitrogen. The β-actin (KC-5A08) and GAPDH (KC-5G5) antibodies were from Kangcheng (Shanghai, China). Melatonin and all other reagents were purchased from Sigma Chemical Co. (St. Louis, Missouri) unless otherwise indicated.
The U87 MG and A172 cell lines were obtained from the Shanghai cell bank and cultured with Dulbecco’s modified Eagle’s medium (DMEM/F12) (Sigma) supplemented with 10% fetal bovine serum (FBS) (GIBCO, Grand Island, New York), 10 units/ml penicillin, and 10 µg/ml streptomycin at 37°C in a humidified 5% CO2 atmosphere. U87 MG-LC3 stable cell lines were generated from cells that had been transfected with the EGFP-LC3 plasmid using Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendations. Stably transfected EGFP-LC3 cells were selected in DMEM/F12 containing 10% FBS and 600 µg/mL G418 (Invitrogen). The LC3 expression level was determined by confocal microscopy.
Immunofluorescence staining was performed as described previously . U87MG and A172 cells were fixed with 4% paraformaldehyde in TBS (Tris Buffered Saline: 0.05 m Tris, 0.9% NaCl, pH 7.6) for 15 min at room temperature, rinsed in TBS (Tris-Buffered Saline: 0.05 m Tris, 0.9% NaCl, pH 7.6) thee times for 10 min each, and were treated with 0.3% hydrogen peroxide in TBS for 30 min to quench endogenous peroxidase activity. We used 1% triton-1000 for permeabilization. The anti-LC3 (1:300) antibodies were added, and the samples were incubated overnight at 4°C.
After being treated with melatonin, agomelatine or rapamycin for 24 hours, the U87 MG cells were treated with 10 μM monodansylcadaverine, 20 nM Mito Tracker Red and 75 nM LysoTracker Red for 30 min, respectively. All imaging was performed on a Zeiss LMS710 confocal microscope, and the data were processed as described previously .
Western blot analysis
The U87MG and A172 cells were homogenized in radioimmunoprecipitation assay (RIPA) buffer [50 mM Tris-HCl (pH 7.4), 0.1% SDS, 1% Triton X-100, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, and 1 mM Na3VO4] containing 1 mM phenylmethylsulfonyl fluoride (Amresco, Solon, Ohio, USA) and protease inhibitor cocktail (Roche, Indianapolis, IN, USA). The protein samples were separated on a 12% SDS polyacrylamide gel and detected with antibodies against LC3, Beclin-1, Bax, Bcl-2, phospho-mTOR/mTOR, phospho-4E-BP1/4E-BP1, phospho-p85S6K/p85S6K, or phospho- p70S6K/p70S6K. β-actin (KC-5A08, Kangcheng, China) or GAPDH (KC-5G5, Kangcheng, China) were used as a loading control, and then the blots were probed with appropriate HRP-conjugated secondary antibodies. The protein bands were detected by enhanced chemiluminescence (ECL) (Amersham Bioscience, UK) and quantified using the Image J software program (NIH).
Flow cytometric analysis of apoptosis
The U87MG or A172 cells were seeded in 6-well plates at a density of 1×106 cells/well. The cells were treated for 24 hours with 1 mM melatonin or 10 mM 3-MA. Afterwards, the cells were washed in PBS and stained with FITC-conjugated Annexin V and propidium iodide (BD Biosciences) in binding buffer. The cells were incubated at room temperature for 15 min in the dark and analyzed by a Beckman Coulter FC500 flow cytometer (Beckton Dickinson, Franklin Lakes, NJ, USA) using the WinMDI2.9 software program.
The statistical analysis was performed with the SPSS software program (SPSS Software, Chicago, IL, USA). The differences between the groups were evaluated by a two-tailed Student’s t-test or a one-way ANOVA, as appropriate. Throughout this study, the values are expressed as the means ± SEM. Values of P < 0.05 were considered to be significant.