Human embryonic kidney cell line 293T (HEK293T) was purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in DMEM medium supplemented with 10% FBS, 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin at 37°C, 5% CO2 -95% O2.
The SAG1 recombinants were kindly given by Dr. Na Yang (Shenyang Agricultural University). Transfection was performed according to the instruction of Polyfectine (PEI) transfection reagent (Biowit Technologies, China). Confluent HEK293 cells cultured in a 60 cm2 dish were transfected with 10 μg recombinant plasmid plus 20 μl Polyfectine(PEI) transfection reagent. The transfection efficiency was determined by western blot after 48 hours of transfection. The recombinant plasmids we used were pEGFP-N1, pEGFP-N1-SAG1-GT1, pcDNA3.1 (+)-Flag-SAG1-GT1, pcDNA3.1 (+)-Flag-SAG1-RH, pcDNA3.1 (+)-HA-RACK1, pcDNA3.1 (+)-mCherry-RACK1. The SAG1 positive cells were collected by FACS sorting of HEK293 cells transfected with EGFP-SAG1 recombinant plasmid and amplified under normal culture condition. Cells transfected with pEGFP-N1 were also sorted and used as control.
HEK293 cells transfected with Flag tagged SAG1 from GT1 or RH strains were collected after 48 hours of transfection. The expression of Flag-SAG1 from GT1 or RH strains were determined using western blot. The anti-Flag immunoprecipitation was performed and the precipitates were subjected to Mass Spectrum analysis and the possible binding proteins of SAG1 were identified.
Preparation of whole cell lysates and western blot analysis was performed as described. The primary antibodies used in this study were anti-EGFP (1:1000, Origene, USA), anti-HA (1:1000, Origene, USA), anti-Flag (1:1000, Santa Cruz, USA), anti-LC3 (1:1000, Cell signaling, USA), anti-RACK1 (1:1000, Cell signaling, USA) and anti-Actin (1:1000, Santa Cruz, USA).
Flow cytometry analysis of canonical and non-canonical autophagy
Flow cytometry analysis of canonical and non-canonical autophagy were performed as previously described . Briefly, HEK293 cells were co-transfected using Flag/EGFP-LC3 or Flag-SAG1/EGFP-LC3. After 48 hours, Cells were treated with baflomycin A (BAF1) or hydroxychloroquine (HCQ) for 2 hours. Control cells (CON) were treated with DMSO. The mean fluorescent intensity (MFI) was detected using flow cytometry. MFIs of HCQ or BAF1 treated cells represents total autophagy and canonical autophagy level respectively. Canonical autophagy was referred as MFIBAF1-MFIcon/MFIHCQ-MFIcon and noncanonical autophagy as 1-MFIBAF1- MFIcon/MFIHCQ- MFIcon.
Cell lysates contain 1000 μg of total protein from HEK293 cells co-transfected with Flag-SAG1 and HA-RACK1 were pre-cleared with 50 μl of protein A-Sepharose beads(Santa Cruz, USA) for 1 h at 4°C and incubated with 5 μg of anti-Flag antibody overnight at 4°C on a rotator. The total volume of this reaction was 1ml. Following antibody incubation, 100 μl of protein A sepharose beads (50% slurry) were added and rotated at 4°C for 3 hours. The beads were then centrifuged at 12,000g for 3 minutes and washed for 3 times with 1% NP40 lysis buffer. The precipitates were eluted by adding of 50 μl of 1× SDS-PAGE sample loading buffer (50 mm Tris-HCl, pH 6.8, 100 mm DTT, 2% SDS, 0.1% bromphenol blue, 10% glycerol), followed by boiling at 100°C for 5 min. The supernatant obtained after centrifugation was resolved by 10% SDS-PAGE and subjected to Western blot analysis using anti-HA antibody.
GST pulldown assay
Whole cell lysates containing 1000 μg of total protein were incubated with GST tagged SAG1 protein from GT1 strain or GST (negative control) overnight at 4°C on a rotator(20 μg each), subsequentially add 50 μl of glutathione-Sepharose 4B (50% slurry) (GE Healthcare, USA) 3 hours at 4°C on a rotator. The resins were then washed 5 times with ice-cold lysis buffer. Binding Proteins were eluted by boiling for 5 min and centrifuged for 3 min at 12,000 g. The supernatant was resolved by SDS-PAGE and subjected to Western blot analysis using anti-RACK1 antibody as detecting antibody.
Confluent HEK293 cells were co-transfected with pEGFP-N1-SAG1and pcDNA3.1 (+)-mCherry-RACK1 using PEI transfection reagent in a 35 cm2 culture dish. The transfection system were as follows: 4 μg of pEGFP-N1-SAG1 and 8 μg of mCherry-RACK1 plus 20 μl of PEI. After 48 hours of transfection, the distribution of SAG1 and RACK1 was observed by fluorescent microscope.
Total RNA was isolated from HEK293 cells transfected with Flag-SAG1. The levels of IL-1β, IL-6, IL-12, and TNF-α mRNA were determined by qPCR. The primers we used were as previously reported , .
Senescence-Associated β-Galactosidase Stain
Cellular senescence-associated β-galactosidase staining was conducted using a senescence β-galactosidase staining kit (Beyotime, Shanghai, China) following the manufacturer’s instruction. Briefly, Cells in a 6-well culture plate were washed with ice cold PBS and fixed with fixing solution for 30 minutes. After being washed with PBS 3 times, 1 ml of staining solution was added to each well and incubated at 37°C overnight. The staining status was observed and photographed using an inverted microscope.
MTT analysis was performed as previously reported. Briefly, Cells were seeded in a 96-well plate (5000 cells/well) and cultured for 48 h, subsequently 50 μl MTT (0.5 mg/ml) (3-(4, 5 dimethylthiazol-2-yl)-2, 5-di-phenyltetrazolium bromide, Sigma, USA) was added to each well and incubated for another 2 h. The dye was dissolved with DMSO and the absorbance was measured at 570nm using a Microplate Reader.
All data are presented as mean ± standard deviation (SD). Each experiment was carried out at least in triplicates. Two-tailed student's t-test was performed to analyze the difference between two groups and one way ANOVA was used to evaluate the difference of multiple groups. A value of p < 0.05 was considered statistically significant.