Primary cell cultures
Cortical neuronal cultures were prepared from whole cerebral cortices of C57BL/6 mouse embryos (E16). Brain tissue was diced into small fragments and incubated in 0.25% trypsin and 200 μg/ml DNase I in PBS. The suspension was then filtered and centrifuged. The pellet was resuspended in PBS and recentrifuged, and after a final wash in feeding medium, the cells were plated into T75 flasks coated with polyornithine (10 μg/ml). The plating density was 80 million cells in 25 ml of medium. To obtain neuron-enriched cultures, cells in the flasks were treated with at least three cycles of 25 μM cytosine arabinoside (2 d on, 3 d off) to kill dividing astrocytes. The feeding medium during this time was minimum essential medium supplemented with 10% fetal bovine serum, nonessential amino acids, 1 mM sodium pyruvate, 2 mM glutamine, and 10% dextrose. All culture medium supplies were from Invitrogen (Burlington, Ontario, Canada). The resulting cells after three cycles of cytosine arabinoside treatment were neurons in excess of 90% purity, with astrocytes, microglia, and precursor cells forming the rest. The neuron-enriched cultures were retrypsinized and plated at 100,000 cells/well in 16-well Lab-tek slides (Nunc, Naperville, IL) in the above medium. Purity of neuronal cultures was> 95% as confirmed by random staining with neuronal and glia markers. 5 days after plating, neurons had developed a dense network of extensions.
Antibodies and reagents
The adenovirus of the GFP-LC3B fusion protein (C3007) was obtained from Beyotime Institute of Biotechnology. Some chemical reagents were purchased from Sigma, including 3-methyladenine (3-MA, M9281), bafilomycin A1 (Baf A1, B1793), thapsigargin (Thap, T9033), AO (A8097), MDC (30432) and carbobenzoxy-valyl-alanyl-aspartyl- [O-met hyl]- fluoromethyl ketone (Z-VAD-FMK, V116); antibodies against autophagy-related protein 5 (ATG5, WH0009140m1), and MAP1LC3B (L7543), were also obtained from Sigma. The antibody against beclin1 (BECN1, 612112) and ACTA (10731) was obtained from BD Transduction Laboratories and Santa Cruz Biotechnology. Other antibodies against poly(ADP-ribose) polymerase 1 (PARP1, 9542), p-EIF2S1 (9721), ATF4 (11815) and DDIT3 (3087) were obtained from Cell Signaling Technology.
Neurons were cultured in Earle's minimal essential medium (Sigma, M0275) containing 10% fetal bovine serum (Gibco, #10099-141) and 100 U/mL penicillin/streptomycin (Thermo Scientific, #15140148) in a 5% CO2 incubator at 37 °C. Neurons were treated with either vehicle or hemin (100 μM; Sigma, St. Louis, MO, USA) for 0~6 hs.
Transmission electron microscopy
Neurons were collected and fixed in a solution containing 2.5% glutaraldehyde in 0.1 M sodium cacodylate for 2 hrs, postfixed with 1% OsO4 for 1.5 hrs, washed and stained in 3% aqueous uranyl acetate for 1h. The samples were then washed again, dehydrated with a graded alcohol series, and embedded in Epon-Araldite resin (Canemco, #034). Ultrathin sections were cut on a Reichert ultramicrotome, counterstained with 0.3% lead citrate and examined on a Philips EM420 electron microscope.
GFP-LC3 puncta formation assays
Neurons were infected with GFP-LC3B adenovirus (MOI=100:1) for 24 hs, then cultured with either vehicle or hemin (100 μM) for 24 hs, and fixed in 4% paraformaldehyde for 10 minutes at 37 °C. Confocal microscopy was performed with a Radiance 2000 laser scanning confocal microscope (Bio-Rad, San Francisco, CA), followed by image analysis with LaserSharp 2000 software (Bio-Rad, San Francisco, CA). Images were acquired in a sequential scanning mode. According to methods for monitoring GFP-LC3 puncta formation assays, the average number of MAP1LC3B puncta per cell in GFP-MAP1LC3B-positive cells was determined.
Western blot analysis
Cell medium was removed and plates were washed three times with chilled PBS. The cells were quickly scraped and collected by centrifugation at 4°C, then stored at -80°C. Cell samples were sonicated with Western blot lysis buffer. Protein concentration was determined using a Bio-Rad protein assay kit (Hercules, CA, USA). A 15 μg portion of protein from each sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a hybond-C-pure nitrocellulose membrane (Amersham, Piscataway, NJ, USA). Membranes were blocked in Carnation nonfat milk and probed with the primary and secondary antibodies. Rabbit polyclonal antibody was used at a 1:1000 dilution (Cappel, MP Biomedicals Inc., OH, U.S.A). Detection was accomplished with goat anti-rabbit IgG (Bio-Rad, Hercules, CA, USA; 1:2500 dilution). The antigen-antibody complexes were observed with the ECL chemiluminescence system (Amersham, Piscataway, NJ, USA). Membranes were stripped and reprobed with antibody against actin (Sigma, St Louis, MO, USA; 1:6000 dilution). The relative densities of bands were analyzed with the NIH Image (Version 1.61).
According to the manufacturer’s instructions (Sigma), Cell viability was determined with an MTT assay. Following treatment, neurons were incubated with MTT at a final concentration of 5 mg/L for 2 hours and then dissolved in the MTT solubilization solution. The cell survival rate was measured with an absorbance at 590 nm (A590) by a microplate reader (Bio-Rad).
Cell death assay
Cell death was assessed using a PI staining assay. The cells were trypsinized, collected, and resuspended in 2 ml of PBS, then incubated with the PI staining solution at 37 °C for 30 min in the dark before being finally measured with flow cytometry.
The ratio of apoptotic cells was evaluated by staining 5×105 cells with an ANXA5/annexin V-FITC/PI Detection Kit (Invitrogen, V13242) according to the manufacturer’s protocol. The samples were analyzed by flow cytometry (BD FACScan Flow cytometer, United States) to determine the percentage of cells displaying annexin V+/PI- (early apoptosis) or annexin V+/PI+ staining (late apoptosis). For each sample, we report the percentage values corresponding to annexin V-FITC positive cells. Three independent experiments were performed for each assay condition.
Acridine orange staining
In acridine orange-stained cells, the cytoplasm and nucleus appear bright green and dim red, respectively, and acidic compartments appear bright red. The intensity of the red fluorescence is proportional to the degree of acidity. After receiving the specified treatments, cells were incubated with acridine orange solution (1 mg/ml) for 15 min in drug-free medium at 37 °C and washed with PBS. Then, cells were trypsinised and analysed by flow cytometry using a FACScan cytometer and CellQuest software. Statistical analyses were performed as described above.
Monodansylcadaverine (MDC) staining
Monodansylcadaverine (MDC) staining was used to quantify the induction of autophagy with heme. Following treatment, cells were stained with MDC at a final concentration of 10 mM for 10 min at 37 °C, collected and fixed in 3% paraformaldehyde in phosphate-buffered saline for 30 min. The cells were then trypsinised and analysed by flow cytometry using a FACScan cytometer and CellQuest software. For each condition, the percentage of cells with characteristic punctuate MDC staining indicative of autophagy was assessed.
The results are expressed as the mean ± standard error (SEM). Two group data were analyzed by Student’s t-test, and multiple group data were analyzed using one-way analysis of variance (ANOVA). Statistically significant differences are indicated by asterisks (*P<0.05).