Cell Culture and Treatment
We followed the methods of Gu Yanting et al. 2013 (11). Human Embryonic Kidney 293 cell (HEK293) cells are used in this experiment which are kept in the Central Laboratory of Aviation General Hospital. HEK293 cells are a cell line derived from human embryonic kidney cells, which have the characteristics of high transfection efficiency and easy culture. HEK 293 cells are considered to be an in vitro model of adrenal cells, rather than typical kidney cells.
We followed the methods of Yan Wang et al. 2019. Cells were plated into 96-well plates and incubated for 24 h to allow cell adherence. Then, the culture medium was replaced with DMEM. After 24 h of incubation, cell viability was determined by colorimetry using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Insoluble formazan crystals were dissolved in dimethyl sulfoxide (DMSO) and measured at 490 nm with a Thermo microplate reader (Thermo Fisher Scientific, Waltham, MA, United States).
We followed the methods of Hanahan. 1983 (12). Rat SIRT1 cDNA fragments was then subcloned into pcDNA3.1/V5-His-A vector to form recombinant plasmids DNA (rSIRT1). HEK293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) (GIBCO) containing 10% fetal bovine serum (FBS) (Life Technology), 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C with 5% CO2. One day before transfection, HEK293 cells were seeded in 6-well plates at a concentration of 3.0 × 105 per well. Recombinant plasmid DNA and control vector was transfected into the cells, respectively. Transfection was conducted using FuGENE HD Transfection Reagent (Promega, Madison, USA). Epifluorescence inverted microscopy was employed to detect the transfection efficiency at 24 h, 48 h and 72 h. After 72 hrs of transfection, 80% of 293T cells were able to express GFP with 2μg DNA. Cells from each group were collected at 72 h for mRNA and protein analyses.
HEK293 cells were transfected with nontargeting small interfering RNA (siRNA) and siRNA targeting SIRT1 (purchased from GenePharma, Shanghai, China) using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, United States) according to the manufacturer’s protocol. Cells were then divided into three groups: rSIRT1, rSIRT1+ siRNA and rSIRT1+Control siRNA. The rSIRT1 cDNA (0.8μg) alone or combined with siRNA (3.2μg) was mixed in 50μl antibiotic-free DMEM to generate transfection complexes. The siRNA targeting human SIRT1 sequences were used: sense, 5′-CCCUGUAAAGCUUUCAGAAdtdt-3′, and antisense, 5′-UUCUGAAAGCUUUACAGGGdtdt-3′ . After cotransfection of SIRT1 cDNA and SIRT1 siRNA, HEK293 cells were seeded in 6-well plates at approximately 40% con- fluence and incubated at 37 °C for 24 h.
The generation of intracellular ROS was examined by flow cytometry using the oxidation-sensitive probe, 2,7-dichlorofluorescein diacetate (DCFH-DA) (Applygen Co. Ltd., China). Cells (3.0×105) were incubated with 10 mM DCFH-DA in complete medium for 30 min at 37℃ to allow cellular incorporation. The cells were then washed twice softly in 1 ml of PBS. Single cells were then analyzed by fluorescent inverted microscope (OLYMPUS). A total of 10,000 cells were counted per sample.
Western Blot Analysis
Protein lysates (20–50 μg) of cells were separated in 12% sodium dodecyl sulfate gels and then were transferred onto polyvinylidene fluoride membranes. After blocking in 5% BSA, membranes were incubated with specific primary antibodies, including rabbit V5 (1:5000; Cell Signaling Technology, Danvers, MA, United States), rabbit TGF-β1 (1:3000; Cell Signaling Technology, Danvers, MA, United States), rabbit Smad3 (1:1000; Cell Signaling Technology, Danvers, MA, United States), rabbit SIRT1 (1:1000; Cell Signaling Technology, Danvers, MA, United States), rabbit P53 (1:2000; Cell Signaling Technology, Danvers, MA, United States), rabbit mTOR (1:1000; Cell Signaling Technology, Danvers, MA, United States), rabbit p-mTOR (1:1000; Cell Signaling Technology, Danvers, MA, United States), rabbit LC3 antibody (1:2000; Sigma-Aldrich, St. Louis, MO, United States) and mouse β-actin antibody (1:3000; Santa Cruz Biotechnology, Santa Cruz, CA, United States) overnight at 4◦C. After washing with TBST, the membranes were incubated with horseradish peroxidase-conjugated IgG. Blots were developed using ECL Detection Kit (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom) and then detected using a ChemiDoc XRS system (Bio-Rad, Hercules, CA, United States). Protein bands were quantified using densitometry with Image J (NIH, Bethesda, MD, United States).
Quantitative data were presented as the mean ± standard error. Comparisons among groups were performed using one-way analysis of variance, and GraphPad Prism software version 6.0 (GraphPad Prism, San Diego, CA, United States) was used for the analysis. P < 0.05 and <0.01 were considered statistically significant.