2.1 Cell lines and cell culture
Human adenoid cystic carcinoma cell(ACC-83) was kindly provided by Peking university. The cells were grown at 37℃ atmosphere with 5% CO2 and 95% air. RPMI 1640 medium supplied with 1% penicillin/streptomycin (HyClone) and 10% fetal bovine serum (FBS) (BI,Biological Industries) were used to culture the ACC-83 cells . the medium was changed every 2 days.
2.2 CCK-8 assay
According to the manufacturer’s instructions, the cell counting kit-8 (Dojindo)was used to measure ACC-83 cells proliferation. ACC-83 cells were seeded to 96-well culture plates(Corning) with 1×10^4 cells in every well. After incubation for 24h with 5% CO2 and 95% air at 37℃,the plates were treated with different concentration proteasome inhibitor MG132 which 2.5,5,10,40,70,100μM for 3,6,9,12,18h.Zero setting was the group that without ACC-83 cells. Group incubated with complete 1640 medium was used as the control group. Every plate was set three times. Premix the CCk-8 and serum-free 1640 medium with the proportion of 1:10.After remove the original complete 1640 medium,100μL premixed solution was added to each well, the cultures were incubated at 37℃ for 2 h. Spectrophotometer(Thermo, Finland) was used to measure The optical density (OD) of each well at a wavelength of 450nm.The concentration of 40μM MG132 was considered to be better choice for this experiments.
2.3 Western blot analysis
The western blot analysis will prove the protein expression changes of Nrf2/Keap1 pathway and P62 after ACC-83 cells incubation under different concentrations of MG132. ACC-83 cells were rinsed twice with cold phosphate-buffered saline (PBS)(Hyclone).And then lysed on ice with RIPA buffer(Solarbio) containing protease inhibitor cocktail with the proportion of 100:1 for 5min.The lysed cells were collected and centrifuged at 12,000rpm for 15 min at 4℃.The protein concentrations in whole cell lysates were determined using BCA protein assay kits(Solarbio).The bovine serum albumin was used as a standard. A total of 30 μg protein was calculated by the volume of lysates for each sample. And equal amount of protein were loaded on a 10-12% SDS-PAGE gel (Beyotime) .Gel electrophoresis was performed at 40 V for 30 min and the following is 100V for 1h.Then transferred the separated polypeptides to a 0.45-μm polyvinylidene difluoride (PVDF) membrane at the condition that 220mA for 30-100min.The PVDF membranes were blocked in a 5% non-fat milk-TBST solution(10 mm Tris-HCl, pH 8.0; 150 mm NaCl; 0.05% Tween-20) for at least 60 min at room temperature while shaking. Then washed the membrane 5 times for 7min each time with TBS-0.05% Tween-20(TBST). Subsequently incubated overnight at 4℃ with primary antibodies against Nrf2 (abcam)and Keap1 (proteintech)and P62(cst, Cell Signaling Technology).After washed 3 times with TBST the membranes were incubated with peroxidase-conjugated secondary antibodies(proteintech)for 60min with shaking. GAPDH(1: 1000 dilution, proteintech)was detected in the same membrane to ensure equal protein loading. Protein bands were visualized by Amersham Imager 600.
2.4 Quantitative real-time polymerase chain reaction (RT-PCR)
ACC-83 cells were seeded in 6-well plates at a density of 1×105 cells/well, and incubated with 1640 as the control, MG132(10、40、70uM)as trial group for 12h. Total RNA from ACC-83 cells after incubated with MG132 was extracted using RNAiso reagent(takara) according to the manufacturer’s instructions. First-strand cDNA was synthesized using PrimeScript™ RT Reagent kit Reverse Transcription System. Real-time PCR was performed with a Roche Light Cycler 480 device in a reacting system that total volume of 20 µl. The cycling parameters used were: 95°C for 30 s, followed by 40 cycles of 95°C for 5 s, 60°C for 30 s, and 72°C for 30 s, and a dissociation program of 95°C for 15 s, 60°C for 30 s, and 95°C for 15 s. Every independent experiment was performed in triplicate.
2.5 Flow cytometry
ACC-83 cells were seeded in six-well plates with a density of 6X10^4 cells per well and incubated for 24 h. The cells were treated with MG132 at concentrations of 10, 40, or 70 μM. Conditions respectively for 12 h. The detection of cell apoptosis was through the Annexin V-FITC apoptosis detection kit. The ACC-83 cells were harvested and rinsed twice with cold PBS, trypsinized, and then washed twice by PBS . resuspended in binding buffer and collected into FACS tubes. The standard protocol tubes were successively stained with Annexin V-FITC (5μL) and PI (5μL), and the other tubes stained with both Annexin V-FITC (5μL) and PI (5 μL) as the experimental groups. And then analyzed with flow cytometry.
2.6 Cell immunofluorescence
ACC-83 cells were seeded to a 24-well plate with the coverslip. Treated with proteasome inhibitor MG132 for 24h when the cell reached a confluence of 60~70 % per plate. The cell washed twice by PBS and fixed with 4% paraformaldehyde solution for 1 hour in the indoor temperature, following rinsed thrice by PBS. And then permeabilized and blocked with PBS which containing 0.5% Triton X-100 and blocked with 1 % bovine serum albumin PBS for 20 minutes at RT. Rinsed trice by PBS the same with the former. Cells were incubated with primary antibody against Nrf2 (cst, Cell Signaling Technology)and Anti-SQSTM1/p62 both at a dilution of 1:100 at 4 °C overnight. After PBS washed trice the 488(abcam)and 594(abcam)secondary antibodies were incubated the same conditions with primary antibody for 1 hour in the 37℃. Cell washed thrice with PBS. Following DAPI solution was added. Before imaging cells were rinsed thrice for 5 min per wash. Fluorescence images were captured by a Leica digital microscope. Images were merged using Adobe Photoshop CS6, and no other modifications were made.
2.7 Statistical analysis
All analyses were performed using graphpad prism 8.0 software. Differences between groups were analyzed using two-tailed Student's t-test. In all of the analyses, P<0.05 was considered to indicate a statistically significant difference.