Cell line and culture.
The CML cell line (K562) was purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. The cells were grown in PRMI 1640 medium (Gibco; Thermo Fisher Scientific, Inc.) and contained 10% foetal bovine serum and 1% penicillin/streptomycin (Gibco; Thermo Fisher Scientific, Inc.). The cells were cultured in a saturated humidified incubator with 5% CO2 at 37℃.
A transient transfection cell line with UVRAG-Knockdown and a mitochondrial autophagy model was established.
Transfection of cells with siRNA was used to knock down the UVRAG gene, and a scrambled shRNA was used as a negative control. CCCP, a mitophagy inducer, was used to induce mitophagy. Cells in the logarithmic growth phase were seeded in a 6-well plate. The transfection method was carried out in strict accordance with Lipofectamine 3000 specifications. Then, cells were incubated at 37℃ with 5% CO2. After 24 or 48 hours, cells were collected for qPCR or Western blot.
Detecting cell proliferation by CCK-8 assay.
K562 cells in the logarithmic growth phase were seeded in a 96-well plate (1 × 104 cells/well), and different concentrations of CCCP (0, 10, 20, 50, 100 µmol/L) were added. The proliferation rate was detected by CCK-8 assay according to the manufacturer's instructions (Vazyme Biotech Co., Ltd). Ten ul CCK-8 reagent was added to each well after 0, 6, 12, 24, and 48 hours of cell culture. Absorbance at 450 nm was measured after incubation for two hours at 37℃.
K562 cells in the logarithmic growth phase were collected and seeded in a 6-well plate (1 × 104 cells/well), which were divided into the NC group, the UVRAG siRNA group, the UVRAG siRNA + CCCP group, and the CCCP group. Cells in the UVRAG siRNA group and the UVRAG siRNA + CCCP group were transfected with siRNA. After 36 hours, cells in the UVRAG siRNA + CCCP group and the CCCP group were treated with CCCP, and the final concentration of CCCP was 50 µmol/L. After continuous culture for 12 hours, cells were collected for detection.
Detection of mitochondrial mass and mitochondrial membrane potential (MMP) by flow cytometry.
Cells in each group were resuspended in 0.5 ml medium, and 0.5 ml JC solution was added. All were mixed fully and incubated in a 37℃ incubator for 20 minutes. During cell incubation, JC-1 dyeing buffer (1×) was prepared, then cells were centrifuged at 600 g and 4℃ for four minutes. The supernatant was discarded. Cells were washed with the dyeing buffer twice. Next, 300 µl JC-1 dyeing buffer (1×) was added to each group. MMP was detected by flow cytometry. Cells were resuspended in a 12-well plate when mitochondrial mass was detected. Five µmmol/L NAO fluorescence probe was added to cells in each group and incubated at 37℃ for 30 minutes and washed with PBS twice to remove the remaining fluorescent probes that did not enter the cells. Mitochondrial mass was detected by flow cytometry.
Detection of ROS by flow cytometry.
According to the manufacturer's instructions, ROS was detected by the Reactive Oxygen Species Assay Kit (Shanghai ZCIBIO Technology Co., Ltd). All cells were harvested and added with 500 µl DCFH-DA diluted to 10 µmol/L in a serum-free medium. They were then incubated for two hours at 37℃ and washed with PBS three times. All samples were placed under a flow detection.
Verification of the transfection effect of UVRAG gene by qPCR.
Cells were washed with PBS, and TRIzol reagent (Vazyme Biotech Co., Ltd.) was used to extract total RNA according to the manufacturer's instructions. A spectrophotometer was used to detect RNA concentrations of 1500 ng/µl and A260 nm/A280 nm between 1.8–2.0. Subsequently, the PrimeScript RTPCR Kit (Vazyme Biotech Co., Ltd.) was used to synthesise cDNA, and the reaction conditions were as follows: 37℃, 15 minutes, and 80℃, five seconds. RT-qPCR was performed using a One-Step SYBR PrimeScript RT-qPCR Kit (Yeasen Biotechnology (Shanghai) Co., Ltd.) with the following thermocycling conditions: 95℃ for five minutes, 95℃ for ten seconds, 55–60℃ for 20 seconds, 72℃ for 20 seconds, a total of 40 cycles. Relative expression was calculated by the 2 - △△CT method.
Cells were lysed with a lysis buffer (KeyGEN BioTECH) for 30 minutes on ice. The supernatant was collected by centrifugation at 14,000 rpm for 30 minutes at 4℃. The BCA protein assay kit was used to detect protein concentrations. SDS-PAGE: protein (50 µg/lane) were separated using a 10% SDS polyacrylamide gel, then transferred to PVDF membranes. The membranes were blocked with 5% skimmed milk for two hours at room temperature and incubated overnight at 4℃ with the anti-UVRAG antibody (dilution, 1:1,000; rabbit YN1925; Immunoway), the antiLC3 antibody (dilution, 1:1,000; rabbit 12741; CST), the antiP62 antibody (dilution, 1:1,000; rabbit 8025; CST), the antiBNIP3L antibody (dilution, 1:1,000; rabbit YN2077; Immunoway) and the antiβ-actin antibody (dilution, 1:1,000; mouse 60008; Immunoway). They were then incubated with HPR Goat Anti-Rabbit IgG (dilution, 1:5,000; 40295; Immunoway) for one hour at room temperature. Densitometry was performed using Image J.
Statistical analysis was performed using GraphPad Prism 7.0 software (GraphPad Software, Inc.). The data were presented as the mean ± SEM. The statistical comparison between the two groups was performed using Student's t‑test. P < 0.05 was considered to indicate a statistically significant difference.