2.1 Reagent and antibody
DEX and DFO were purchased from sigma (Missouri, USA). Dulbecco's modified basic medium (DMEM), sugar free DMEM, fetal bovine serum (FBS), phosphate buffered saline (PBS) and trypsin EDTA were purchased from GIBCO (New York, USA). The primary antibody against β-actin (CL594-66009) and LCN2 (26991-1-AP) were purchased from Proteintech (Chicago, Illinois). The primary antibodies against p62 (#A19700), Beclin-1(#A7353), LC3B (#A19665), MMP9 (#A0289), Bax (#A19684), Bcl-2 (#A19693) and NF-KB (#A19653) were purchased from Abclonal (Wuhan, China). The plasmids of LCN2 was designed by Kaiji Biological Company (Nanjing, China). Mir-326-5p mimic and mir-326-5p inhibitor were designed and constructed by Jima Biological Company (Shanghai, China).
2.2 Cell culture
H9C2 cells were purchased from Sciences Cell Bank of Chinese Academy (Shanghai, China). The cells were cultured in DMEM containing 10% fetal bovine serum (FBS) and 1% antibiotics at 37 ° C in a humidified incubator with 5% CO2. Cell morphology was evaluated using an inverted microscope by the ZEISS system (ZEISS, USA).
2.3 Establishment of OGD/R model
The model was established with reference to the literature and slightly modified.
DMEM was cleared and the 96-well plate was washed by PBS for three times. Sugar free DMEM without FBS was added instead. Then the plate was placed in anaerobic bag (Mitsubishi, Japan) at 37°C in humidified incubator for 3, 6, 9 and 12 hours. When the scheduled time is reached, we discarded sugar free DMEM and washed the 96-well plate with PBS for three times. Complete DMEM with 10% FBS was added and the cells was cultured in normal condition for 1, 3, 6 and 9 hours.
2.4 Plasmid transfection
According to Lipofectamine 2000 instructions (Thermo Fisher, USA), H9C2 cells were transfected with corresponding siRNA or miRNA plasmid.
2.5. Real-time PCR
We lysed H9C2 cells with Trizol reagent (Life Technologies, MA, USA). Then we precipitated the total RNA with isopropanol (Aladdin, Shanghai, China) and transcribed to cDNA according to the instructions of HifairII 1st Strand cDNA Synthesis SuperMix (YEASEN, Shanghai, China). Finally, we performed real-time PCR with Hieff™ qreal-time PCR SYBR Green Master Mix (Low Rox Plus) (YEASEN, Shanghai, China) in Applied Biosystems 7500 machine (Thermo Fisher, MA, USA) following the manufacturer's guidelines.
2.7. Western Blot
Protein of each sample was extracted and added with the same account. Then it was separated by SDS-PAGE and transferred to PVDF membrane. After blocking with 5% BSA, the PVDF membrane was incubated with corresponding primary antibody overnight. HRP bound secondary anti-rabbit was applied for 2 hours next. The PVDF membrane was added with ECL (YEASEN, Shanghai, China) and protein stripe was analyzed by Image Lab software.
2.8 Immunofluorescence assay
H9C2 cells were inoculated on the cover glass in 24-well plate. After clearing DMEM, the cell morphology was fixed with 4% paraformaldehyde for 20 minutes and penetrated by 0.1 % Triton. Then it was blocked by 1% bovine serum albumin for 2 hours and incubated with primary antibody overnight. FITC rabbit secondary antibody was added for 2 hours. Then, 2ul fluorescence quencher was added on the cover glass. All images were taken blindly with Zeiss microscope (Germany).
2.9 Malondialdehyde (MDA) assays
According to the instructions, H9C2 cells were intervened with MDA working solution from Jiancheng Bioengineering Institute (Nanjing, China). MDA concentration was determined at 532 nm by microplate reader.
2.10 Iron ion detection
The Fe2+ level in H9C2 cells was detected using Iron detection kit (ferrozine microplate method) from Yita biotech company (Beijing, China) according to the manufacturer's instructions.
2.11 Mitochondrial membrane potential detection
Mitochondrial membrane potential level was detected using mitochondrial membrane potential test kit (JC-1 method) from Yita biotech company (Beijing, China). Pictures was taken under the fluorescence microscope and fluorescence intensity was measured using Synergy HTX Multi-Mode Microplate Reader (Agilent, USA) with excitation/emission wavelength of 490 nm/530 nm and 525 nm/590 nm.
2.12 Oxygen free radical (ROS) assay
According to the guidelines, H9C2 cells were intervened with ROS working solution from Jiancheng Bioengineering Institute (Nanjing, China). The fluorescence intensity was measured with excitation/emission wavelength of 488 nm/525 nm.
2.13 Superoxide dismutase (SOD）assay
According to the instructions, H9C2 cells were intervened with SOD working solution from Jiancheng Bioengineering Institute (Nanjing, China). SOD concentration was determined at 450 nm by microplate reader.
2.14 Luciferase assay
H9C2 cells were transfected with mir-326-5p mimics and WT or MUT psiCHECK2-LCN2. After 24 hours, the cells were lysed on ice and added with firefly luciferase detection reagent according to the instructions. Then the samples were measured using Synergy HTX Multi-Mode Microplate Reader.
2.15 Cell Counting Kit-8 (CCK-8) assay
H9C2 cells were plated in 96-well plates for different intervention for 24 hours. Then, 10μl CCK8 reagent was added and incubated at 37°C for 4h. The results were determined by microplate reader at 450 nm.
2.16 MTT Assay
H9C2 cells were plated in 96-well plates for different intervention. 10μl MTT reagent was added and incubated at 37°C for 4h. Then DMSO was added to dissolve the formazan. The results were determined by microplate reader at 592 nm.
Results was expressed as mean ± standard deviation and mapped with graphpad prism 7.0 (graphpad software, San Diego, California). Statistical analysis was determined by one-way ANOVA of Student t-test (comparison between two groups) or student Newman Keuls test (more than two groups). The value of P < 0.05 was considered statistically significant.