R28 cell culture and treatment
R28 cells were purchased from Cell Resources Center, Shanghai Academy of Life Sciences. They were cultured in glucose-free DMEM at 37°C in a 5% CO2 environment, supplemented with 10% fetal bovine serum (FBS), 1×105 U/L penicillin, and 100 mg/L streptomycin until 80% of the cells fused. Cells were then incubated in DMEM medium without FBS for 24 hours. Subsequently, R28 cells were cultured with various concentrations of D-glucose (5.5, 15, 30, and 40 mM) for 12 hours, or with 30 mM D-glucose or 30 mM mannitol for the indicated time (0, 15, 30, 60, 90, 120, and 180 min). The mRNA levels of p66Shc and phosphorylation of p66Shc S36 were detected by Real-time PCR and Western blotting, respectively.
Concurrently, R28 cells were divided into five groups: i. control (CTL) group (untransfected R28 cells + 5.5 mM glucose); ii. high glucose group (untransfected R28 cells + 30mM glucose); iii. high glucose overexpressed p66Shc group (R28 cells transfected with p66Shc expressing plasmid + 30mM glucose); iv. high glucose p66Shc KO group (R28 cells transfected with small interfering RNA for p66Shc + 30mM glucose); v. high glucose S36A mutant group (R28 cell transfected with serine 36 variant p66Shc + 30mM glucose).
Small interfering RNA for p66Shc, p66Shc overexpression plasmid, and mutant p66Shc serine 36 plasmids were all obtained from Biofort Biotechnology Co., LTD (Shanghai, China). They were transfected into the R28 cells according to manufacturer’s instructions.
The production of ROS by R28 cells was evaluated by labeling cells with MitoSox followed by detection by laser confocal microscopy at 2 and 24 hours after glucose stimulation. Flow cytometry was used to detect apoptosis at 48 hours after glucose stimulation.
Real time PCR
We used The Trizol method to extract RNA. P66Shc primer was designed by Primer Premier 5.0 software and synthesized by Invitrogen Biotechnology Co., Ltd. (Shanghai, China). The primer sets are listed in Table 1. We mixed the sample with 1 ml Trizol (Invitrogen Life Technologies, Shanghai, China) and shake it well, then put it on ice for about 5 minutes to denaturate it completely. After that, we mixed the mixture with 0.25 ml chloroform, shake for 15 seconds, and stand for 3 minutes at 4 C, centrifuged the mixture at 12,000 × g for 10 min, and collected the supernatant. Then, we added 0.5 ml isopropanol, mixed, and let the mixture rest for 10 minutes at room temperature, followed by centrifuged the mixture for 10 minutes at 12,000 × g, and discarded the supernatant. After cleaning the precipitated particles, we added 1.5 ml 75% ethanol, centrifuged the mixture for 8 minutes at 12,000 × g. We removed the supernatant, and air-dried the precipitated RNA for 5-10 minutes. The Revert Aid First Strand cDNA Synthesis Kit (Thermo, Shanghai, China) was used to reverse transcribe total RNA (2 µg), according to the manufacturer's protocols. GAPDH was used as the internal reference gene. Amplification of 20 µl reaction mixture was carried out with the following PCR program: 95°C for 10 min, 40 cycles of 95°C for 15 s, and 60°C for 60 s. We used 2-ΔΔCt method to analyze the data.
Western blot
RIPA lysis buffer containing protease inhibitors was used to extract protein. We mixed the protein sample with the sample buffer, heated it in 100°C water for 10 minutes, and then cooled the mixture in ice water. Then we loaded the proteins (10~20 g) onto a sodium dodecyl sulphate (SDS) 12% polyacrylamide gel. After electrophoresis, the protein was transferred onto polyvinylidene fluoride membranes. 5% skimmed milk in 0.1% Tween/Tris-buffered saline (TBST) was used to block the nonspecific binding. Membranes were incubated with primary antibodies against p66Shc (1:1000; Abcam, Shanghai, China), serine 36 phosphorylated p66Shc (1:500; Abcam, Shanghai, China), and GAPDH (1:5000; Boster, Shanghai, China) overnight at 4°C. Then we used phosphate buffered saline containing Tween (PBST) to wash the membrane three times in a shaker. After that, we immersed the membrane in the appropriate horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. We washed the membrane three times again in PBST, then visualized the protein band with enhanced chemiluminescence reaction kit (Goodbio Biological Technology Co., LTD, Wuhan, China), and captured the protein band with Tanon 5500 imager (Tanon, Shanghai, China). We analyzed the band with AlphaEaseFC (Alpha Innotech, San Leandro, CA, USA). Each band is normalized according to the corresponding GAPDH band. The changes of protein expression were expressed as the ratio of levels in diabetic rats to non-diabetic rats.
MitoSOX Red
The production of ROS by R28 cells was evaluated by labeling cells with MitoSox (Invitrogen) according to the manufacturer instructions. MitoSOX Red is a kind of reactive cell permeable dye. It can target mitochondria rapidly and selectively, and is mainly used to observe mitochondrial O2 levels. MitoSOX red fluorescence (excitation at 510 nm and emission at 585 nm) was detected by laser scanning confocal microscopy equipped with a bandpass filter (Zeiss LSM 510; Carl Zeiss, Thornwood, NY, USA). After staining with MitoSOX Red, mitochondrial O2 was also detected by flow cytometry.
Flow cytometry
We used an annexin V-FITC (fluorescein isothiocyanate) Kit (Dojindo, Shanghai, China) to quantify the percentage of undergoing apoptotic cells. Briefly, R28 cells were divided into the groups as above-mentioned. Then the cells were inoculated into 6-well plates. After 48 hours of treatment, the cells were digested by trypsin, then centrifuged at 1000 rpm at 4 C for 5 minutes, washed by PBS, and incubated with a mixture of 5 µl annexin V-FITC conjugate and 5 µl propidium iodide (PI) at room temperature in darkness for 15 minutes. A flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA) was used to detect apoptosis.
Isolation of mitochondria
Mitochondria were prepared by using Thermo Fisher mitochondria isolation Kit (Pierce, Rockford, IL, USA) (19). Briefly, after digesting the homogenized samples with the kit reagents, the samples were centrifuged for 10 minutes at 700 × g, then centrifugation for 15 minutes at 3000 × g. The pellet thus obtained was washed, suspended in PBS, and used for the quantification of p66Shc and cytochrome C in mitochondria.
Mitochondrial DNA damage
Mitochondrial DNA is sensitive to oxidative damage as it lacks repair mechanisms. When the aggregation and replication of mitochondrial DNA are inhibited, the number of long copies of mitochondrial DNA decreases, suggesting mitochondrial DNA damage. Detecting the ratio of long fragments to short fragments of mitochondrial DNA can thus reflect the integrity of mitochondrial DNA (20).
Briefly, long (8.8 kb) and short (223 bp) mtDNA regions were amplified using PCR, and the amplified products were resolved on an agarose gel. Relative amplification was quantified by normalizing the intensity of the long product to the short product (8.8 kb/223 bp).
Statistical analysis
We used the Statistical Package for Social Sciences (version 11.0, SPSS Inc., Chicago, IL, USA) to perform statistical analysis. The mean ± standard deviation (SD) expressed data. The difference between two groups used the Student’s t-test to analyze. The differences among groups used One-way ANOVA to analyze. P <0.05 was considered a significant difference.