DMEM medium, penicillin and streptomycin were purchased from Gibco. FBS was purchased from Zebio Biotechnology Co., Ltd. (Beijing, China). T25 cell culture flasks and cell culture plates were from JET BIOFIL. Invitrogen (Carlsbad, CA) lipofectamin2000 was used. The apoptosis detection, cell cycle detection, and ROS detection kits were purchased from Solibao Technology Co., Ltd. (Beijing, China). JC-1 came from Yeasen Biological Technology Co., Ltd. (Shanghai, China). The caspase-3 activity detection kit was from Beyotime Biotechnology (Shanghai, China). The blood DNA kit was from Kangwei Century Biotechnology Co., Ltd. (Beijing, China). Flow cytometery was conducted on the ACEA Biosciences (San Diego, CA) Novocyte flow cytometer.
2.3 Methods
2.3.1 DNA and mtDNA isolation and Exome Sequencing
After the male proband’s (II-5) family map was built, DNA was extracted from peripheral blood samples of 3 affected family members (Ⅱ-5,Ⅲ-19,Ⅳ-3,) and 2 unaffected family members (Ⅲ-16, Ⅲ-20) using standard methods. However, mtDNA was obtained only from the bone marrow of Ⅱ-5. Oral epithelial cells were collected for normal tissue comparison in mtDNA sequencing test.
DNA was isolated from peripheral blood using a DNA Isolation Kit, and DNA libraries were prepared with KAPA Library Preparation Kit following the manufacturer’s instructions. Array capture hybridization of pooled libraries using capture probes and removal of non-hybridized library molecules were completed using the Agilent SureSelectXT2 Target Enrichment System. Sample dilution, flowcell loading and sequencing were performed according to the Illumina specifications. DNA libraries were sequenced on the Illumina Novaseq platform as paired-end 200-bp reads.
2.3.2 293 T cells transfection and culture for experiments
The cDNA of Six1 was inserted into pRK7-N-Flag between XbaI and EcoRI to construct the vector: pRK7-conl. The c.374A was mutated to c.374G in the pRK7-conl vector to obtain pRK7-case using the Takara MutanBEST kit. 293 T cells, which were serially transfected with pRK7-conl and pRK7-case, were cultured in an incubator containing 5% CO2 at 37°C.
2.3.3 Cell proliferation experiment
Cells in the logarithmic growth phase were collected and trypsinized conventionally, washed with PBS, pelleted by centrifugation, resuspended and counted. The cells were seeded on 96-well plates at 2×103 cells/well at a volume of 100 µl per well. After 12, 24 or 48 h of incubation, 10 µl of CCK-8 solution was added to each well. After 2h of incubation, the absorbance of each well was measured with a microplate reader at a wavelength of 450 nm and the mean was calculated for each group. These results were then analyzed and recorded.
2.3.4 Apoptosis detection
At corresponding time points after transfection, cells were trypsinized, collected, and apoptosis was detected using flow cytometry according to the following steps. The cells were grouped according to the conditions of each group, harvested, and centrifuged at 1000 rpm at 4℃ for 5 minutes to collect the cells. Cells were then, washed twice with pre-chilled PBS, centrifuged at 1000 rpm at 4℃ for 5 min, and 5×105 cells were collected. The PBS was aspirated and cells were resuspended in 100uL of 1X Binding Buffer. Next, cells were stained with 5uL of Annexin V-FITC and 10uL of propidium iodide (PI) Staining Solution and mixed gently. These cells were stained at room temperature for 10-15min in the dark. Then, 400uL 1X Binding Buffer was added, mixed well, and samples were placed on ice. Apoptosis detected by flow cytometry within 1 hour after samples were put on ice.
2.3.5 Cell cycle stage analysis
1×105 transfected cells were added to each tube, 1mL of pre-chilled 1×PBS (pH 7.2–7.3) was added, mixed well, and centrifuged at 1000rpm for 5min to wash the cells. Next, the 1X PBS was aspirated and 500 uL pre-cooled 70% ethanol was slowly added, and mixed by pipetting gently. Cells were fixed at 4℃ for 16h and washed again. Next, 200uL of pre-chilled 1X PBS was added with 2uL of RNase and mixed by gently pipetting. These cells were incubated at 37℃ for 30min to remove RNA. 5ul of PI staining solution was added and the cells were incubated at room temperature for 30min in the dark. Finally, 100uL of this solution was taken (around 1×104 ~ 1×105 cells) and the cell cycle stage was examined by flow cytometry.
2.3.6 Caspase-3 activity test
Cells were collected at the corresponding time point after transfection, trypsinized, collected, and washed once in PBS. After aspirating the supernatant, lysing solution was added at a ratio of 100uL/2 million cells, the pellet was resuspended, and lysed for 15 minutes in an ice bath. The lysate was then centrifuged at 3500 rpm at 4℃for 10–15 min. The supernatant was collected and Ac-DEVD-pNA (2mM) was added, mixed, and solution was incubated at 37℃for 60–120 min. Samples were measured with an ELISA reader at an absorbance of 405nm.
2.3.7 ROS staining
Cells were collected at the proper time point, the medium was discarded, and the cells were washed three times with 1 ml of PBS buffer. 1ml of 1:1000 diluted fluorescent probe DCFH-DA was added and the cells were incubated at 37 degrees for 30 min. Then cells were washed three times with 1ml of PBS buffer, counterstained with DAPI, and washed another 3 times. A confocal laser scanning microscope was used to take pictures to record the distribution and expression of green fluorescence (ROS).
2.3.8 JC-1 detects mitochondrial membrane potential
Cells were collected at the corresponding time point after transfection, trypsinized, harvested, added to JC-1 the staining working solution, mixed and incubated. 1X JC-1 buffer was created according to the kit instructions. After incubation, the supernatant was aspirated, washed three times with 1X JC-1 buffer, and the pellet was collected by centrifugation. The proportion of the JC-1 polymer or monomer was determined using flow cytometry.
2.4 Western blotting test
Cells were harvested and total protein was collected after lysing the cells. The protein electrophoresis was run on 10% SDS-PAGE gels. 20µg of each protein sample was loaded, concentrated at 80 V for 20 min and separated at 120 V for 1 h. Electrophoresis was stopped as soon as the bromophenol blue color marker had run off. The samples were then transferred to PVDF at 110V for 100 min at 4℃ by using the wet blotting method.
Each immunoblot was blocked in 5% nonfat milk in TBST for 2h prior to being incubated overnight at 4℃ with primary antibodies against Six1, Bcl-2, Bax, cleaved-caspase 3 or β-tubulin. Then the membrane was washed 3 times with TBST for 10 min, and then incubated with diluted secondary antibodies in blocking solution for 2h at room temperature. After the secondary antibody solution was fully washed away, PTG ECL chemiluminescence detection kit was used for color rendering, then the blot was transferred to the X-ray film for exposure and analysis.
2.5 Statistical analysis
The experimental results were statistically analyzed using SPSS 19.0 and GraphPad Prism 8.0. The results were expressed as mean ± standard deviation and analyzed by ANOVA test. P < 0.05 indicated that the difference was statistically significant.