Cell culture

A glial cell line, SHG-44, was purchased from Shanghai GeneChem Co. Ltd. (Shanghai, China). The cells were cultured in 96-well plates at densities about 10,000 cells per well in GibcoDulbecco’s Modified Eagle Media (DMEM/F-12; Thermo Fisher Scientific) containing 10% fetal bovine serum (FBS, Thermo Fisher Scientific) supplemented with 1% penicillin/streptomycin (Thermo Fisher Scientific), at 37℃ in a humidified atmosphere with 5% CO2 in the air. The media was changed 2–3 times weekly and at 24 h before the cells were collected.

Establishment of CIRP overexpression on SHG-44 cells

The recombinant lentiviral vector system containing the green fluorescent protein (GFP) reporter used in this study was purchased from Shanghai Genechem Co., Ltd. The vector encoding full length of CIRP gene (AB000362) was constructed, and PCR and DNA sequencing confirmed the accurate insertion of the CIRP cDNA. For transfection, the glial cells were plated in 96-well plates and transfected with either with CIRP lentiviral vectors (CIRP overexpression group, OE-CIRP) or with the GFP lentiviral vectors (negative GFP control group, N-GFP) in serum-free medium for 12 h at a multiplicity of infection (MOI) of 40. Polybrene (5ug/mL) was added to enhance the infection efficiency. The other cells without lentiviral infection served as a control group (control group, CON) and the cells transfected with naïve lentivirus served as a mock group (mock group, MOCK). The cells were then washed and embedded into complete medium. Four days after transfection, the GFP gene expression was examined using fluorescence microscopy. The transfection efficiency was calculated.

Cell treatment

The OE-CIRP cells and MOCK cells were challenged with four injuries including oxidation, hypoxia, glucose deprivation and glutamate induced neurotoxicity. First, the MOCK cells were treated with various concentrations of H2O2, CoCl2 and glutamate respectively for 12 h to choose the suitable concentrations for further procedures. After the chosen concentrations confirmed, four injuries were applied. Oxidative stress was induced by H2O2 as following. The medium was removed firstly, then H2O2 was added on the cells. The medium was added immediately into the wells to get the final chosen concentration of H2O2. This procedure was repeated every 2 h until 12 h to further analysis. CoCl2 (232,696, Sigma-Aldrich) was prepared as aqueous solutions at chosen concentration to induce chemical hypoxia. The glial cells were treated with the CoCl2 solution for 12 h. The cells were cultured with glucose- and serum-free DMEM (11,966,025, Thermo Fisher Scientific) for 12 h to achieve glucose deprivation. The cells were exposed to glutamate at chosen concentration for 12 h to induce neurotoxicity.

MTT assay for cell viability

Before performing MTT assay, the culture medium was replaced with 100 µL of fresh one. MTT solution (10 µL, 12 mM, Vybrant™ MTT Cell Proliferation Assay Kit, Thermo Fisher Scientific) was added to each well, including a negative control of 10 µL of the MTT solution added to 100 µL of medium alone. After incubating at 37 °C for 4 h, the cell viability was analyzed with a microplate reader (Fluoroskan Ascent, Thermo Fisher Scientific), absorbance at 570 nm.

Real-time polymerase chain reaction

Total RNA was isolated, and cDNA was synthesized as we reported previously [1]. The primer sets used in this study were designed and provided by Sangon biotech, Shanghai China and listed in Table 1. Real-time reverse transcriptase (RT-PCR) analysis was performed using an SLAN Real-Time PCR System (Hongshi, Shanghai, China). PCR conditions used for amplification were as follows: initial denaturation at 95 °C for 5 min, 40 cycles of denaturation at 95 °C for 15 s, annealing at 65 °C for 30 s, and elongation at 84 °C for 30 s. Data were collected in real time at the end of each cycle. Negative controls, without reverse transcriptase, and water controls were included in each reaction. In addition to real-time and melting curve analysis of the reactions, amplified products were separated electrophoretically on 2% agarose gels with ethidium bromide and visualized under UV light to confirm proper amplicon size, as well as the absence of non-specific products. All PCR products produced a single specific product.

Table 1 Primer sets used in this study

Relative expression data were calculated according to the two-delta threshold (∆∆Ct) relative quantification method. Normalization of Ct values was performed by subtracting β-actin Ct from the CIRP Ct values acquired for each sample. To calculate the relative difference in the number of cycles between the samples, the average Ct value of the samples from the control group was selected as baseline. This number was subtracted from each of the Ct values that were previously normalized to β-actin. The relative difference between sample groups was calculated based on the difference in Ct values using 2-Δ(ΔCt).

Double immunohistochemistry

The cells were incubated with PBS twice for 10 min each before being fixed with 4% paraformaldehyde at room temperature for 15 min and then washed with PBS containing 0.05% Tween20 to remove the fixative. The fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 min at room temperature and blocked with 1% Blocker BSA for 15 min at room temperature. Anti-CIRP antibody (AB166775, Abcam) and anti-EGF antibody (701,538, Invitrogen) as primary antibodies were added to the respective wells and incubated overnight at 4 °C. After primary antibody incubation, the cells were washed with PBS, and then incubated with Alexa Fluor 488 donkey anti-goat IgG secondary antibody and Alexa Fluor Plus 594 Donkey anti-Rabbit IgG secondary antibody for 2 h at room temperature. The cells were washed with PBS before incubation for 1 min with DAPI nuclear stain (D9542, Merck). Images were taken on an Olympus confocal microscope.

Western blot

The cells was washed twice with cold PBS (0.01 M pH7.2～7.3), resuspended in ice-cold cell lysis buffer (9803, Cell Signaling Technology) with 1mM PMSF (8553, Cell Signaling Technology) and incubated at 4 °C for 30 min. The lysates were centrifuged at 12,000 rpm for 10 min at 4 °C. The protein of the supernatants was stored at − 80 °C. CIRP and EGF levels were analyzed by Western blotting with monoclonal antibody to CIRP ((AB166775, Abcam) and EGF (701,538, Invitrogen). β-Actin was used as the internal control. Blot bands were quantified using an optical scanner and the Labworks system (Labworks Inc, Winnipeg, Manitoba, Canada).

Statistical analysis

Statistical analysis was performed using Sigma Plot (12.5) for Windows. Data were presented as the mean ± SD of three independent experiments. Statistical analysis of differences was carried out by a one-way ANOVA. P < 0.05 was considered to indicate statistical significance.