Reagents and antibodies
GO nanosheet was obtained from Sigma-Aldrich (Shanghai, China). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), streptomycin-penicillin and EDTA/trypsin were purchased from Thermo Fisher Scientific (Shanghai, China). An LDH-Cytotoxicity Assay Kit and a bicinchoninic acid (BCA) protein assay kit were from Beyotime (Nanjing, China). A cell counting kit-8 (CCK-8) test was purchased from Dojindo Molecular Technologies (Shanghai, China). Rapamycin and carbonyl cyanide 3-chlorophenylhydrazone; carbonyl cyanide 3-chlorophenylhydrazone; carbonyl cyanide m-chlorophenylhydrazone (CCCP) were from MCE (Shanghai, China). 2- (4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) staining solution was obtained from Leagene Biotechnology (Beijing, China). 2.5% glutaraldehyde for electron microscopy was obtained from Nacalai Tesque, INC (Japan). Lyso-Tracker Red and RIPA lysate buffer were obtained from Solarbio Life Sciences (Beijing, China). A human cathepsin B activity assay kit was purchased from Cusabio (Wuhan, China). An Annexin V-FITC-PI Apoptosis Kit and a Mitochondrial Membrane Potential (MMP) assay kit (JC-1) were obtained from KeyGEN BioTECH (Jiangsu, China). Rapamycin was purchased from HefeiAnqi Biotech Co., Ltd. (Hefei, China). Halt protease inhibitor and phosphatase inhibitor cocktail was from La Roche Ltd. (USA). Polyvinylidene fluoride (PVDF) membranes were purchased from R&D Systems (USA). Primary antibodies, including antibodies for LC3I/II, p62, PI3K, Akt/p-Akt, mTOR/p-mTOR, Bax, Bcl-2, caspase-3, and cleaved caspase-3, and horseradish peroxidase-conjugated (HRP) secondary antibodies were obtained from Abcam (Shanghai, China). Chemiluminescence substrate was obtained from EMD Millipore (Massachusetts, USA).
Characterization of GO NPs
The microstructure of GO was observed by scanning electron microscopy (SEM; Hitachi Ltd., Japan). Moreover, an atomic force microscope (AFM; Agilent Inc., USA) and a transmission electron microscope (TEM; Hitachi Ltd, Japan) for the original characterization of GO.
The functional groups and molecular structure of GO were determined by Raman spectroscopy (Renishaw, UK) and Fourier-transform infrared spectroscopy (FTIR) (Thermo Fisher Inc., USA). Basic chemical bonds and element content of GO were detected using X-ray photoelectron spectroscopy (XPS; Kratos, Japan).
GO nanosheets were sonicated using an ultrasonic processor (Biosafer, China) in pure water and cell culture medium to obtain uniformly dispersed suspension. The hydrodynamic size and zeta potentials of GO NPs in solution with uniform dispersion were measured using a Nano Zetasizer (Malvern Panalytical Ltd., England).
Cell culture and preparation of GO suspension
The rat astroglioma-derived F98 cell line (ATCC CRL-2397) was purchased from Zhongqiao Xinzhou Co., Ltd. (Shanghai, China). After recovery from liquid nitrogen, F98 was cultured in DMEM containing 10% FBS and 1% streptomycin-penicillin and incubated in 95% humidified air with 5% carbon dioxide at 37°C to obtain a culture density of 1000 cm-2. During the culture process, the growth status and cellular morphology were observed using an ICX41 inverted biomicroscope (Sunny Optical Ltd, China). The cell culture medium was replaced every other day, and the average subculture time was 3-4 d.
A GO suspension with a concentration of 1 mg/mL was obtained in advance as the stock solution. After irradiation with 60Co, GO nanosheets were sonicated for 1 h 8-times (60 Hz) by an ultrasonic processor. Before each administration, the storage solution of GO was diluted to different concentrations with complete medium supplemented with 10% FBS and then ultrasonicated again for 30 minutes with a high-power ultrasonic crusher.
Cell viability assay
F98 cells were first inoculated in 96-well plates (Corning, USA) at a density of 5×103 cells/well and cultured in a 37°C incubator for 24 h prior to GO treatment. Then, the cells were exposed to fresh GO suspension at final concentrations of 10, 20, 40, 50, 60, 80, and 100 μg/mL, while the control group was left untreated. After 6, 12 and 24 h of treatment, cell proliferation activity was evaluated by the CCK-8 test according to the supplier’s protocol.
Cellular viability was further estimated by measuring the LDH release from injured cells. Briefly, the F98 cells were inoculated in 96-well plates (5×103 cells/well) and cocultured with GO suspension at different concentrations (20, 40, 50, 60 and 80 μg/mL) for 6 and 24 h. Wells containing only culture medium without cells (background blank control) and wells containing cells without GO (sample control) were used as negative controls. For the positive control, the maximum release of LDH was triggered by LDH release agent. After treatment, all supernatants were transferred to new 96-well plates and assessed by the LDH assay kit according to the supplier’s protocol. At a wavelength of 490 nm, the absorbance was analyzed using a full-spectrum microplate reader (Reagen, China).
On the basis of the CCK-8 cell viability test, 40, 60 and 80 μg/mL GO were selected, and rapamycin (100 nM) was added for the same amount of time in the rescue experiment. Similarly, we measured cell viability by CCK-8 and LDH kits.
SEM and TEM observation
F98 cells were seeded on coverslips in 12-well plates (5×104 cells/well) and cocultured with or without GO (60 μg/mL) for 24 h. Thereafter, the cells were fasted overnight with 2.5% glutaraldehyde at 4°C and washed with PBS. Subsequently, 1% osmium tetroxide was added to treat the cells at 4°C for 1 h. Finally, the cell coverslips were dehydrated with graded ethanol and gold sputtered. The cells were observed and photographed with a Hitachi S-3400N SEM.
For TEM observations, F98 cells were seeded in 6-well plates (105 cells/well) and then cocultured with or without GO (60 μg/mL) for 24 h. The cells were washed with PBS, harvested with trypsin, centrifuged at 800 rpm for 5 minutes, and then fasted with 2.5% glutaraldehyde for 50 minutes at room temperature and for 3 h at 4℃. After dehydration with a graded series of ethanol, the cells were embedded with epoxy resin at 90°C for 3 d. Finally, ultrathin sections (70 nm) were dyed with uranyl acetate-lead citrate and then observed and photographed by TEM.
Detection of autophagy flux formation by immunofluorescence staining
As previously described, F98 cells were seeded on glass coverslips and co-exposed with or without GO (60 μg/mL) for 24 h. Cells were also treated for the same amount of time with rapamycin (100 nM) as the positive control. Thereafter, the cells were washed with PBS and fasted with 4% paraformaldehyde for 9 minutes. Then, the cells were blocked for 1 h, cocultured with the primary antibody at 4°C overnight, and finally cocultured with the secondary antibody for 45 minutes at room temperature. The cells were dyed with DAPI staining solution at room temperature for 5 minutes. Images were immediately observed by a BX63-AFM microscope (Olympus, Japan). FITC and DAPI excitation/emission filters were selected for photography.
Detection of lysosomal acidity
F98 cells were seeded and treated with 40, 60 and 80 μg/mL GO for 24 h. After incubation, Lyso-Tracker Red was diluted to a working concentration with complete medium at a ratio of 1:16 000 and then cultured with cells at 37℃ for 1 h. The cells were then washed with PBS. After removing the excess water with filter paper, the cells were inverted on a carrier plate, dripped with anti-fluorescence quenching tablet sealer, and observed by a BX63-AFM microscope. A FITC excitation/emission filter (Ex= 495 nm, Em= 515 nm) was selected for photography. To quantitatively evaluate the strength of FITC fluorescence, cells were harvested with trypsin after the same treatment and then resuspended in PBS and instantly analyzed by flow cytometry (BD Biosciences, USA).
Cathepsin B activity assay
Cells were inoculated in 9 cm culture dishes at a density of 9×105 cells/dish. Similarly, F98 cells were treated with 40, 60 and 80 μg/mL GO for 24 h. The cells were subsequently washed with precooled PBS and harvested with trypsin. After two cycles of repeated freezing and thawing, the debris and cell fragments were centrifuged at 5000 rcf for 5 minutes to collect the supernatant liquid, and cathepsin B activity was detected according to the supplier’s protocol.
Annexin V-FITC-PI assay
F98 cells were plated on 6-well plates (2×105 cells/well) and treated with 40, 60 and 80 μg/mL GO for 24 h. In the rescue experiment, 60 μg/mL GO and 60 μg/mL GO-100 nM rapamycin were included as the experimental groups. A blank control group and a rapamycin control group were also included. The old culture medium was abandoned, and the cells were washed with PBS. After that, the cells were harvested with trypsin and washed again with PBS. Then, 500 μL of binding buffer was added to the resuspended cells, and 5 μL of Annexin V-FITC and 5 μL of propidium iodide (PI) were added in turn. After incubation in the dark at room temperature for 15 minutes, the percentages of apoptotic cells were immediately detected by flow cytometry.
Nuclear staining with DAPI
Cells were cultured overnight on polylysine-coated dishes and then treated with 0, 40, 60 and 80 μg/mL GO for 24 h. Cells were treated separately with CCCP (50 μM) for 30 minutes as a positive control. After incubation, the cells were harvested with trypsin, subsequently washed with PBS, fasted with 4% paraformaldehyde for 30 minutes, and then labeled with DAPI staining solution for 10 minutes. Changes in the nuclei were instantly observed by a BX63-AFM microscope. A DAPI excitation/emission filter (Ex= 364 nm, Em= 454 nm) was selected for photography.
Assessment of MMP
Similarly, after 24 h of exposure to 40, 60 and 80 μg/mL GO, the cells were washed with PBS and incubated at 37°C for 20 minutes with 5 µg/mL JC-1, a fluorescent carbocyanine dyestuff widely used to detect MMP. The cells were then washed and resuspended in JC-1 assay buffer (1X), and red-green fluorescence was immediately detected by flow cytometry.
After 24 h of treatment with 40, 60 and 80 μg/mL GO, the cells were washed with PBS, and RIPA lysate buffer containing protease inhibitor and phosphatase inhibitor cocktail were prepared to digest the cells. Cell granules and lysates were collected and centrifuged at 12000 rpm for 30 minutes. The supernatant was saved, and the total protein content was measured by a BCA protein assay kit. The newly extracted protein was used in 10% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and then transferred to polyvinylidene fluoride (PVDF) membranes. Subsequently, the membranes were blocked for an hour and then incubated overnight with primary antibody at 4°C. The primary antibodies included LC3I/II, p62, PI3K, Akt/p-Akt, mTOR/p-mTOR, Bax, Bcl-2, caspase-3, and cleaved caspase-3. Thereafter, the membranes were incubated with HRP-conjugated secondary antibody at room temperature for an hour. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was designated as a loading control and showed no significant differences among groups. Finally, the protein bands were activated by chemiluminescent substrate and visualized by an enhanced chemiluminescence (CL) system (Tanon Science & Technology Co., Ltd, China). The band intensities were quantified using ImageJ.
In the rescue experiment, 60 μg/mL GO and 60 μg/mL GO-100 nM rapamycin were included as the experimental groups. A blank control group and a rapamycin control group were also included. The primary antibodies were LC3I/II, p62, Bax, Bcl-2, caspase-3, and cleaved caspase-3, and the same operational steps were used as described above.
The results are expressed as the mean ± standard deviation (SD) values of three independent experiments. All analyses were carried out using GraphPad Prism (USA) with unpaired Student’s t-test or one-way ANOVA. The data were considered to be statistically significant when the probability (p) < 0.05.