Cell and reagent
Cell and reagent HT22 cells (mouse hippocampal neuron cell line), donated by Professor Weilin Jin laboratory, Shanghai Jiaotong University. Fetal bovine serum, (from South America Lonsera company); DMEM/F-12 cell culture medium( American Hyclone company); 0.25% trypsin digestive juice(from Gibco company); Glutamic acid powder (No. 09581), Hoechst 33342 live cell dye (No. B2261), (from Sigma Inc., USA); Tenovin-1 (No. S8000, p53 specific activator); Erastin (No, S7242, Iron death-specific inducer, from Selleck Corporation), p53 antibody (No, 10442-1-AP, from Wuhan Sanying Corporation), 4-HNE (No, ab46545), xCT (No, ab175186, antibody, from Abcam Company, UK);β-actin, GAPDH antibody (from Santa Cruz Company, USA); FITC-labeled goat anti-rabbit antibody(from Beijing Zhongshanjinqiao Biotechnology Co., Ltd.); DHE fluorescent probe, cell proliferation test kit (cell counting kit-8,CCK-8, from Biyuntian Institute of Biotechnology); ECL light-emitting reagent kit; BODIPY™ 581/591C11 lipid oxidation probe (from Thermo Fisher, USA); FeRhoNox™-1 iron ion probe (from Goryochemical Corporation, Japan)
Instrument fluorescence microscope (Leica Corporation, Germany); cell incubator (Thermo Corporation, America); enzyme labeling (Tecan Corporation, Germany); centrifuge (Beckman Corporation, Germany); protein electrophoresis, Bioshine ChemiQ chemiluminescence imaging system (Bio-Rad Corporation, America).
HT22 cells were randomly divided into 6 groups : (1) Control group(CON) : HT22 cells were cultured in DMEM/F12 medium; (2) p53 acitvation group (P53): HT22 cells were pretreated with 1 µ mol/L Tenovin-1 for 6 h, and HT22 cells were accompanied with p53 activation. (3) Glutamate exposure group (Glu): HT22 cells were treated with Glutamate with final concentration of 5 mmol/L; (4) Erastin treated group (Era): HT22 cells were treated with 0.5 µmol/L Erastin; (5) Glutamate exposure after p53 activation group(p53-H): After HT22 cells were adminitrated by 1 µmol/L Tenovin-1 for 6 h, and they were exposed to 5 mmol/L Glutamate. (6) Erastin-treated group (Era-p53): HT22 cells were pretreated with Tenovin-1 at the final concentration of 1 µmol/L for 6 h, and then they were administrated with 0.5 µmol/L Erastin.
Total RNAs was quantified by the NanoDrop ND-2000 (Thermo Scientific) and the RNAs integrity was assessed using Agilent Bioanalyzer 2100 (Agilent Technologies).The sample labeling, microarray hybridization and washing were performed based on the manufacturer’s standard protocols. Briefly, total RNAs were transcribed to double strand cDNAs and then synthesized cRNAs. Next, 2nd cycle cDNAs were synthesized from cRNAs. Followed fragmentation and biotin labeling, the 2nd cycle cDNAs were hybridized onto the microarray. After washing and staining, the arrays were scanned by the Affymetrix Scanner 3000 (Affymetrix).
Detection of HT22 cells viability
The viability of HT22 cells was detected by CCK-8 assay. HT22 cells were cultured in 96-well plates at about 1×104/ well for 12 h. Glutamate (final concentration was 1, 5, 10 mmol/Land Erastin (0.25, 0.5, 11 µmol/L were added. The control group and blank group (corresponding amount of cell culture medium and CCK-8 solution) were set up, each hole was set up 5 compound pores. After 6 hours of incubation in incubator, 10 µL CCK-8 solution was added to each well(100 µL medium per well). After incubated with 2 h in the incubator, the absorbance value of each well was measured at 450 nm wavelength by spectrophotometer, and the relative survival rate of each well was calculated. The relative survival rate = (experimental group - blank group) / (control group - blank group), the experiment repeated 3 times.
The protective effects of p53 activation on glutamate damage and iron death induced by erastin in HT22 cells.
The protective effects of p53 activation on glutamate damage and iron death induced by erastin in HT22 cells were detected by PI/Hoechst fluorescence double labeling method. HT22 cells were inoculated with 5×104 cells/well in 24-well plate and treated according to above-mentioned method. After treatment for 8 h, the final concentration of 5 mg/L Hoechst 33342 and PI solution were added to the medium. The cells were incubated with 37℃ and 5% CO2 for 10 min, and the relative cell survival rate was calculated under fluorescence microscope. Relative cell survival rate was expressed as the ratio of Hoechst /(Hoechst++PI+) greater than 500 cells. The experiment was repeated three times.
Western blot was used to detect the difference of p53 and xCT protein expression.
Western blot was used to detect the difference of p53 and xCT protein expression. 1 ×105cells / well were vaccinated into 6-well plate for 12 h, then treated with different concentrations of Tenovin-1 (0.1, 1, 10µmol/L) for 6 h, and then the samples were prepared, and the samples were treated with different concentrations of p53 (0.1, 1, 10 µmol/L) for 6 h. The cells were washed twice by PBS and added with lysate containing protease inhibitors and phosphatase inhibitors. The cells were lysed on ice for 10 min, using a cell brush and scraped off the cells and placed in a 1.5 mL EP tube with ultrasound for 3 times for 5 s. BCA protein quantitative kit was used to detect the concentration of protein. The sample was mixed with 3×SDS buffer at 2:1 volume, boiled at 100℃for 5 min, quickly placed on ice to room temperature and centrifuged at 13000 × g for 5 min. After electrophoresis with 35 µg protein sample per well, the protein was transferred to the PVDF membrane at a constant current of 200 mA for 1 h, which was washed 3 times with 5% skimmed milk powder for 1 h. TBST was at room temperature, β-actin antibody was diluted at a dose of 1:1000 each time at room temperature for 1 h and 4℃overnight. After TBS washing 3 times, and 10 min each time. The cells were incubated at room temperature with 1:5 000 dilution for 1 h, developed with ECL kit and β-actin as internal reference; the expression level of xCT protein was detected: 1×10 5 cells per well were inoculated in 6-well plate for 12 h, and the cells were cultured for 12 h. After 8 hours of treatment, the samples were prepared according to the above method, and the expression level of xCT protein was detected by Western blot. XCT and GAPDH antibody were diluted by 1: 1 000.
The lipid oxidation product 4-HNE was detected by immunofluorescence assay.
The HT22 cells were seeded with 5×10 4 cells per well in 24-well plate. Control, P53, Glutamate and Glutamate-p53 group was selected for 8 h according to above-mentioned method. The cells were washed twice with PBS, fixed at room temperature with 4% paraformaldehyde for 15 min, washed 3 times with PBS, sealed with 15% donkey serum at room temperature for 1 h, washed with PBS for 3 times, and added with an antibody 4-HNE. The dilution ratio is 1:200, 4℃ overnight after incubation at room temperature for 1 h, washed for 3 times, added with second antibody (dilution ratio of 1:400), incubated at room temperature for 1 h, washed for 3 times, then sealed with 10 µg/ml DAPI glycerol, The average fluorescence intensity of different groups was observed under fluorescence microscope.
BODIPY 581/591 C11 lipid oxidation fluorescence probe was used to detect intracellular lipid oxidation
BODIPY 581/591 C11 lipid oxidation fluorescence probe was used to detect intracellular lipid oxidation in HT22 cells inoculated in 24-well plate with 5×104/well. Control, P53, Glutamate and Glutamate-p53 group was selected for 8 h according to above-mentioned method. Methanol dissolved the BODIPY 581/591 C11 fluorescence probe to the storage solution with the concentration of 10 mmol/L, and added the final concentration of 1 µmol/L BODIPY 581/591 C11 fluorescence probe to the treated cells. The cells were incubated at 37℃ and incubated with a fluorescent probe of 1µmol/L BODIPY 581/591 C11. After incubating with PBS for 30 min, the unbound dyes were washed twice with PBS. The fluorescence effect was observed by fluorescence microscope, and the average fluorescence intensity of different groups was observed.
FeRhoNox-1 fluorescent probe was used to detect the changes of intracellular iron ions
FeRhoNox-1 fluorescent probe was used to detect the changes of intracellular iron ions in HT22 cells, which were vaccinated with 5×10 4 cells per well in 24-well plate. Control, P53, Glutamate and Glutamate-p53 group was selected for 8 hours according to the above-mentioned method. The FeRhoNox-1 fluorescent probe was dissolved into 1 mmol/L storage solution by DMSO. After being treated with 24-well plate, the cells were washed twice with PBS and diluted with HBSS solution to 5 µmol/L working solution. 200 µ L was added to each well. The working solution was incubated in 37℃, 5% CO2 incubator for 1 hour, washed twice with HBSS solution and observed under fluorescence microscope. The average fluorescence intensity of each group was calculated.
The statistical analysis was carried out with SPSS 20.0.0 software. The measurement data were expressed as the mean ± SD from at least three biological replicates, and the comparisons between groups was performed by one-way ANOVA (Analysis of Variance), pairwise comparison using Tukey test. A value of p < 0.05 was considered statistically significant. Meanwhile, affymetrix GeneChip Command Console (version 4.0, Affymetrix) software was used to extract raw data. Next, Expression Console (version1.3.1, Affymetrix) software offered RMA normalization for both gene and exon level analysis. Then the gene expression analysis and alternative splice analysis proceeded separately. ① Gene expression analysis: Genesrping software (version 13.1; Agilent Technologies) was employed to finish the basic analysis. Differentially expressed genes were then identified through fold change as well as P value calculated with t-test. The threshold set for up- and down-regulated genes was a fold change > = 2.0 and a P value < = 0.05. Afterwards, GO analysis and KEGG analysis were applied to determine the roles of these differentially expressed mRNAs played in these GO terms or pathways. Finally, Hierarchical Clustering was performed to display the distinguishable genes' expression pattern among samples. ② Alternative splice analysis: Alternative splice analysis was conducted by Transcriptome Analysis Console (version1.0, Affymetrix). Differential exon or junction identified through splicing index as well as P value calculated with One-Way Between-Subject ANOVA (Unpaired). The threshold was splicing index > = 2.0 or < = -2.0