The mouse retinal ganglion cell line (RGC-5) was contributed by Department of Ophthalmology, Second Hospital of Ji Lin University in China . RGC-5 cells grew in Dulbecco’s Modified Eagle Medium (DMEM) (HyClone Laboratories, Inc. UT) and supplemented with 10% fetal bovine serum (FBS, HyClone Laboratories, Inc. UT), 100 U/ml of penicillin and 100 μg/ml of streptomycin (HyClone Laboratories, Inc. UT). The RGC-5 cells used in the experiment were within 2-3 passages post-thawed to minimize the variability in the assays based on our observations. The density of RGC-5 cells was around 80% in 6 ml culture media in 50 ml flask before EHP (elevated hydrostatic pressure).
The cells cultured in the flasks were harvested and RNA was isolated using TRIzol (Invitrogen, Carlsbad, CA). cDNA was synthesized using Thermoscript (Invitrogen, Carlsbad, CA) from 1 μg of total RNA. Each primer pair (β-actin: forward primer
reverse primer 5′-ACTTTTATTGGTCTCAAGTCAGTGTACAG-3′.
NGF: forward primer 5′-AGAACCGTACACAGATAGCAA-3′
reverse primer 5′-TTAATGTTCACCTCGCCCAG-3′.
BDNF: forward primer 5′-AAAACCATAAGGACGCGGACTT-3′
reverse primer 5′-AAAGAGCAGAGGAGGCTCCAA-3′.
GFAP: forward primer 5′-ACGCAATTCCATTTTACCTG-3′
reverse primer 5′-AGGCCATAACTCATGCAAC-3′) was designed to cross at least one intron and was specific to the gene of interest. Negative control was used diluted water for instead. Oligonucleotide primers that recognized the housekeeping gene β-actin were used as normalized control. PCR program consisted of 94°C for 3 min, 36 cycles of 94°C for 45 s, 55°C for 50 s, and 72°C for 2 min, with a final extension at 72°C for 10 min.
The coverslips with fixed cells were washed in 0.01 M PBS for 3 min, incubated in 5% BSA and then reacted with anti-rabbit calpain antibody (Abcam, ab39170, USA, 1:500) or anti-rabbit Brn3a (Boster, BA1773, China, 1:200) and anti-mouse THY1.1 (Abcam, ab8872, USA, 1:500) overnight. Then coverslips were reacted with Cy2 or Cy3-conjugated donkey anti-rabbit or anti-mouse secondary antibodies at 1:200 (Invitrogen, USA). Counterstaining with DAPI, then covered with an anti-fading mounting medium before microscopic examination. Control RGC-5 incubated simultaneously in a conventional incubator at 37°C.
Cell injury and ALLN or Nec-1 usage
A pressurized incubator was designed to expose the cells to an elevated hydrostatic pressure as described in Ju’s study . After 2 hr exposure in this pressure system, the pressure present in three different values: 100 mmHg, 60 mmHg and 30 mmHg, cells were then moved to conventional culture incubator to recover at each recovery time point (6 hr, 12 hr and 24 hr). ALLN (Merck, Germany) and Nec-1 (Sigma-Aldrich, USA) were dissolved in Dimethyl sulfoxide (DMSO) for storage in 10 mmol/L and 1 mg/mL, which were pretreated in 10 μmol/L for 24 hr before cell injury.
At each recovery time point (6 hr, 12 hr and 24 hr), the coverslips were washed in 0.01 M PBS for 3 min, and incubated in 10 μg/ml PI-dye solution at 37°C. After that, cells were fixed in 4% PF (Paraformaldehyde) and washed in PBS counterstaining with DAPI, and then covered with anti-fading mounting medium before microscopic examination. Control RGC-5 was incubated simultaneously in a conventional incubator at 37°C. Quantitative analyses were conducted using approximately 20 merged images (magnification = 40×) to estimate the frequency of cell necrosis.
At each survival time point, cells were digested by sonication on ice in a digestion buffer [150 mM NaCl, 25 mM Tris–HCl (pH 7.4), 2 mM EDTA, 1.0% Triton X-100, 1.0% sodium deoxycholate, 0.1% SDS] containing a cocktail of protease inhibitors (Sigma, USA). Cell lysates were centrifuged at 10,000 × g for 20 min at 4°C. The supernatants were collected, and protein concentration was determined by Bicinnchoninic acid (BCA) assay (Pierce, USA). A total of 100 μg of protein in 62.5 mM Tris loading buffer (pH 6.8, containing 25% glycerol, 2% SDS, 0.01% bromophenol blue and 5% β-mercaptoethanol, Bio-Rad, USA) was boiled for 10 min, and loaded into each lane of 4-20% linear gradient Tris–HCl ready gel (Bio-Rad, USA). The polypeptides were electrotransferred to Trans-Blot pure nitrocellulose membrane (Bio-Rad, USA). Non-specific binding was blocked with PBS containing 5% nonfat milk (Bio-Rad, USA) and 3% bovine serum albumin (Sigma, USA). Membranes were incubated with tAIF (Santa Cruz biotechnology Inc, SC-113116, USA, 1: 200), β-tubulin (Abcam, ab6046, USA, 1:1000), calpain (Abcam, ab39170, USA, 1:500) or actin (Abcam, ab3280, USA, 1:500) antibodies overnight, and then in HRP-conjugated secondary antibodies (1:20000, Bio-Rad, USA) for 1 hr. Immunoblotting products were visualized with an ECL Plus™ Western Blotting Detection kit according to manufacturer’s instruction (GE Healthcare Life Sci., USA), and images were captured in a Molecular Dynamics Phosphor imager (Nucleo Tech Inc., USA). Western blot bands were measured with Image J (National Institutes of Health, USA) to analyze the integrated density value (IDV). The average IDV values of tAIF or calpain with β-tubulin and actin were compared, and the average relative value was obtained.
The cells attached to the flasks were trypsinized followed by a gentle wash. Resuspending the cells in 200 μl of 1× binding buffer, and then added 5 μl of 20 μg/ml Annexin V and 10 μl of 50 mg/ml PI, incubated at RT for 15 min in the dark. After the cells were washed and analyzed by FACS Calibur (Becton, Dickinson Company, USA). The percentages of cells in each quadrant were analyzed using ModFit software (Verity Software House Topsham, USA). Statistical results of flow cytometry were conducted by calculating the PI+ cells numbers. All the results were repeated for three times.
Calpain activity assay
Calpain activity was determined by cleavage of the substrate Ac-LLY-AFC (Abcam, USA). The attached cell were digested, and then centrifuged for 1 min in a microcentrifuge (10,000 × g) then the supernatant was transferred to a fresh tube, a part of the supernatant were used to measure the protein concentration. The fluorescence was measured after 60 min incubation at 37°C along with the substrate in the reaction buffer. Fluorescence was recorded in a Fluoroskan Ascent Fluorimeter (Labsystems, Eragny-Parc, France), and the final results were expressed as Relative Fluorescent Unit (RFU) per milligram protein of each sample.
Figure panels were assembled by using Photoshop CC. The data were analyzed by using SPSS 19.0 (SPSS, USA). One-way analysis of variance (one-way ANOVA) was performed to test differences in average value between groups. All results were presented as mean ± SD. A value of p < 0.05 was considered statistically significant.