Cell culture
Human retinal pigment epithelial cells ARPE-19 were brought from American Type Culture Collection (ATCC, Manassas, VA, USA). ARPE-19 cells were grown in DMEM/F-12 medium (Gibco, New York, USA) at 37°C in a humidified environment that contained 95% air and 5% CO2 and was supplemented with 10% FBS and 1% streptomycin/penicillin. Cells with a confluence of 80–90% were chosen for culturing and subsequent testing. Subsequently, 400 µM of H2O2 (Sigma-Aldrich, St. Louis, MO, USA) was added to the cells and incubated for 6 h. Finally, experiments were performed with H2O2-treated cells.
Lentiviral infection
Human overexpression of PCSK7 and control sequences were designed by GeneChem (Shanghai, China). Then, overexpression of PCSK7 or control oligonucleotides were subcloned into Lentiviral packaging vector LV-013, containing a green fluorescent protein (GFP) (GeneChem, Shanghai, China) and named PCSK7 and NC. After treatment with H2O2, ARPE-19 cells would be transfected with PCSK7 and NC lentiviral vectors in the presence of 5g/mL polybrene (Sigma-Aldrich, St. Louis, MO, USA), and then selected for 7 days with puromycin (Sigma-Aldrich, St. Louis, MO, USA) and cells resistant to puromycin were isolated for further studies.
Cell Counting Kit 8 (CCK8)
Cells from the H2O2-treated ARPE-19 cells line were transfected with a lentiviral vector before being placed in a 96-well plate at a density of 2000 cells/well. Then, a solution of Cell Counting Kit 8 (CCK8; Beyotime Institute of Biotechnology, Beijing, China) was added and left for 1 to 5 days. At various time points, the OD values were measured at 570 nm using a microplate reader (M2009PR, Tecan infinite, Australia).
Apoptosis Detection
ARPE-19 cells transfected with PCSK7 or NC lentivirus were inoculated in 6-well plates at a density of 2 × 105 cells/well and cultured. Cells were collected and stained in Annexin-V/PE Apoptosis Assay Kit (eBioscience, Thermo Fisher Scientific, Waltham, MA, USA) for 25 minutes at room temperature and protected from light. Stained cells were analyzed for apoptosis using a FACSCanto II flow cytometer (BD Biosciences, New Jersey, USA).
Immunofluorescence
For immunofluorescence analysis, cells were cultured in 6-well plates and fixed in 4% formaldehyde for 30 min and permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) for 10 min. The fixed cells were then blocked with 5% BSA for 30 min at room temperature and incubated overnight at 4°C with antibodies targeting PCSK7 (1:200 dilution; 12044-1-AP, Proteintech, Wuhan, China) or Caspase3 (1:200 dilution; ab208161, Abcam, Cambridge, UK). Then, cells were incubated with secondary antibody Goat Anti-mouse-FITC (S0007, Affinity, Changzhou, China) or Goat Anti-rabbit-Fluor594 (S0006, Affinity, Changzhou, China) for 1 h at 37°C and counterstained with DAPI (Sigma-Aldrich, St. Louis, MO, USA) for 10 min. The cells were then visualized under a Fluorescence microscope (Zeiss, Jena, Germany).
Enzyme-linked immunosorbent assay (ELISA)
The concentrations of glutathione (GSH, BC1175, Solarbio Science, Beijing, China), and Iron (ab83366, Abcam, Cambridge, UK) in cells were measured via respective ELISA kits. The optical density (OD) value at 450 nm was evaluated using microplate reader (Thermo Fisher, Waltham, MA, USA).
JC-1 assay
The mitochondrial membrane potential was detected by JC-1 assay. JC-10 kits (CA1310, Solarbio Science, Beijing, China) 1 ml were supplemented in ARPE-19 cells for 20 min at 37°C and rinsed by PBS. The cells were then visualized under a Fluorescence microscope (Zeiss, Jena, Germany) to analyze the images of the green and red fluorescence intensity in each unit. The intensity ratio of red fluorescence to green fluorescence referred to the mitochondrial membrane potential.
Detection of intracellular ROS accumulation
The cells were transferred to the center of a 35 mm glass-bottomed Petri dish and washed three times with 1× PBS. The samples were fixed in 4% paraformaldehyde in PBS solution for 15 minutes at room temperature. Samples were washed twice in PBS to remove residual paraformaldehyde, permeabilized with 0.5% Triton X-100 in PBS for 15 minutes, and then washed 3 times in 1 × PBS. Cells were incubated with Reactive Oxygen Species Assay Kit (Solarbio Science, Beijing, China) at 37°C for 30 min, washed three times with 1 × PBS, and then restained with DAPI for 5 min at room temperature. The staining was observed under an immunofluorescence microscope (Leica DFC500, Germany).
Real-time quantitative PCR and western blotting
For RT-qPCR, TRIzol (Sigma-Aldrich, St. Louis, MO, USA) was used to extract total RNA of cells. After the integrity was verified by electrophoresis, RNA was reversely transcribed into cDNA by Hiscript QRT supermix for qPCR(+ gDNA WIPER) (Vazyme, Shanghai, China). AceQ qPCR SYBR Green master mix reagent (Vazyme, Shanghai, China) was used for qRT-PCR experiment. The primer sequence as follow: PCSK7: F: 5’-CATTGCCTAGGTATCCGGGT-3’; R: 5’-GGGCTTCTCATGTGGCAATC-3’ and GAPDH: F-5′-ATCCCATCACCATCTTCCAG-3′ and R: 5’-ATGACCTTGCCCACAGCCT-3’.
Relative expression was calculated using the 2-△Ct method. GAPDH were amplified as controls for RNA integrity.
For WB, after protein from cells extraction by total protein extraction kit (Goodbio Technology, Wuhan, China), and resolution on 10% SDS PAGE, PVDF membranes (Bio-Rad, California, USA) were electro-blotted. The membranes were sealed with non-fat milk, cultured with the primary antibodies (shown in Table 1) for one night, and then cleaned with Tris-buffered saline and Tween 20. The secondary antibody was then added and incubated for 1 hour with donkey anti-rabbit or anti-mouse IRDye-conjugated IgG (1:3000, Abcam). The images were then obtained by scanning the blots. With the aid of a very sensitive ECL chemiluminescence detection kit, the tagged protein bands were examined.
Table 1
Gene name | LOT | Manufacturer | Dilution ratios |
---|
PCSK7 | 12044-1-AP | Proteintech | 1:1000 |
ACSL4 | ab155282 | abcam | 1:2000 |
FTH1 | ab75972 | abcam | 1:2000 |
GPX4 | ab125066 | abcam | 1:2000 |
Cytochrome C | ab18738 | abcam | 1:2000 |
COX IV | ab202554 | abcam | 1:2000 |
GAPDH | AF7021 | Affinity | 1:2000 |
Statistical analysis
We have presented the experimental results, which are represented as mean ± SD from a minimum of three separate experiments. Statistical analyses were performed using GraphPad Prims (version 9.5.0) and the student’s t-test was utilized to compare between two groups. The statistical significance was set at P < 0.05.