Reagents
Purified anti-mouse CD28 mAb, anti-mouse CD3 mAb, FITC-conjugated anti-mouse CD4 and PE-conjugated anti-mouse CD69 were purchased from BioLegend (SanDiego, CA, USA). The Seahorse XF Glycolysis Stress Test Kit and Mitochondrial Stress Test Kit were offered by Agilent Technologies (Palo Alto, Calif.). Roswell Park Memorial Institute (RPMI) 1640, fetal bovine serum (FBS) and penicillin/streptomycin were ordered from Gibco (South Logan, UT, USA). DAPI (4′, 6-diamidino-2-phenylindole) and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) were bought from Solarbio (Beijing, China). Enhanced ATP Assay Kit, Mitochondrial Membrane Potential Detection Kit and Reactive Oxygen Detection Kit were supplied by Beyotime Biotechnology (Shanghai, China).
Animals
All mice were kept in specific pathogen-free conditions in an animal resource center at Xinxiang Medical University. All experiments were performed using female mice aged 6–8 weeks and in agreement with the animal care guidelines established by Xinxiang Medical University.
Metabolic Assessments
The OCR/ECAR analysis was performed on the XFe24 Extracellular Flux Analyzer (Agilent). The seahorse instruments were preheated. One day before the assay, 1 mL XF Calibrant buffer was injected into the wells of a 24-well utility plate from Seahorse Bioscience and incubated at 37°C overnight in a non-CO2 incubator. Seahorse XF base medium was used to prepare the test solution and was preheated to 37 ℃ before the test. Cells were seeded into an XFe24 microplate at a density of 5×105 cells/well. When the detection reagent of ECAR/OCR was filled into the relevant injection port of sensor cartridges, the cell microplates were incubated at 37°C in a non-CO2 incubator for 45–60 min. Finally, the cell culture microplate was loaded onto the instrument after calibration. Wave 2.4 software (Agilent) was used to analyze the data.
RNA Isolation and qPCR
Total RNA was isolated from Bcl-3−/− Jurkat cells and normal Jurkat cells using Trizol reagent. Briefly, 2000ng of total RNA was retro-transcribed using RT Master Mix (Takara). Q-PCR analyses were performed using SYBR Green supermix in an Applied Biosystems 7500 System. After normalization of expression of HPRT by the 2−ΔΔCT procedure, relative mRNA level was revealed as the fold change relative to untreated controls. The specific primers used were synthesized by Sangon Biotech. mRNA levels were normalized HPRT. The PCR primer sequences were as follows:
For Bcl-3, 5’-AACCTGCCTACACCCCTATAC-3’(forward)
and 5’-CACCACAGCAATATGGAGAGG-3’(reverse)
and for mTOR, 5’- ACTCGCTTCTATGACCAACTGA-3’(forward)
and 5’-TTTCCATGACAACTGGGTCATTG-3’ (reverse)
and for Raptor, 5’-ACTGGAACCTACCTTTGGCTT-3’ (forward)
and 5’-ACTGTCTTCATCCGATCCTTCA-3’ (reverse)
Western Blot Analysis
The protein from Bcl-3−/− Jurkat cells and normal Jurkat cells for Western blot was extracted to detect the protein expression level. Western blot assays were performed with primary Abs against SREBF1, mTOR, P-AMPK, P-Akt and P-Raptor. Cells were collected and lysed in an ice-cold WB/IP lysis buffer (Beyotime, Shanghai, China), and protease and phosphatase inhibitors were added. Then, after the nuclear proteins were extracted, the cell lysates or immunoprecipitates were separated by SDS-PAGE and transferred to PVDF membranes according to the manufacturer’s instructions. After blocking with 5% skim milk, the membranes were incubated with the primary antibody at 4°C overnight and then with the corresponding secondary antibody for 1 hour at room temperature. The membranes were washed with TBST buffer three times for 10 minutes each. At last, the membrane was put into the instrument for exposure and pictures were taken.
Determination of intracellular ATP content
Jurkat and Bcl-3−/− Jurkat cells were seeded into a 24-well culture plate at a density of 1.2×105 cells/well, and each group was set up with 4 duplicate holes. After 24h, the medium was discarded, and ATP lysate was added to each well to lyse the cells. Cells were centrifuged at the speed of 12000 r/min for 5 minutes. The supernatant was collected from each of the wells and detected with an ATP detection kit. Finally, the ATP concentration of each well was calculated according to the standard curve.
Measurement of ROS Levels
1×106 Jurkat and Bcl-3−/− Jurkat cells were incubated in 24-well plates for 24h to detect reactive oxygen species (ROS), respectively. Subsequently, the cells were stained with DCFH-DA (10 µM) for 20 min and treated with CCCP (10 µM) as a positive control. Finally, after staining, the cells were washed and resuspended with PBS buffer and then analyzed by flow cytometry.
Analysis of Mitochondrial Membrane Potential
The Mitochondrial Assay Kit (Beyotime, Shanghai, China) was used to measure the mitochondrial membrane potential (ΔΨm). 1×106 Jurkat and Bcl-3−/− Jurkat cells were added to 0.5 mL diluted JC-1 staining working buffer and well mixed. The cells were incubated in a 37 ℃ incubator for 20 min and then collected by centrifugation. Next, cells were washed twice with JC-1 (1×) dye buffer, then fixed with an appropriate amount of paraformaldehyde fixing solution. Finally, the nuclei of the cells were stained with DAPI and visualized with confocal laser scanning microscopy (CLSM). In addition, the stained cells also could be directly analyzed by flow cytometry.
Proliferative ability
Jurkat and Bcl-3−/− Jurkat cells were collected and co-incubated with 5 µM CFSE. 5×105 cells/ml was suspended in 1640 medium with 10% FBS, and then 1ml/well cell suspension was added to the 24-well plates. After 3 days, the proliferation was detected by flow cytometry.
Activation ability
Briefly, 5×105 Jurkat or Bcl-3−/− Jurkat cells were added to the 48-well plates precoated with CD3 antibodies at 0.5ml per well. 10 µg/ml anti-mouse CD28 antibody was added and incubated for 24 hours. Then the cells were stained with CD69 antibody, and the expression of CD69 was detected by flow cytometry.
T-cell isolation and stimulation
Spleen from Bcl-3−/− and normal wild-type mice were ground and filtered through 40 µm mesh to obtain a single-cell suspension. The red blood cells were subsequently depleted from the cell suspension with erythrocyte lysate. Following erythrocyte lysis, 5×105 cells/well were inoculated into 48-well plates with a 500 µL system per well. After 24 hours, the cells were collected for ECAR detection or flow cytometry.
Flow Cytometry
Jurkat cell or single-cell suspension from the spleen was washed and initially stained with CD4-FITC and CD69-PE. Then, cells were stored at 4°C in the dark for 30 min and washed with PBS 3 times. Activated T cells were identified by CD4+CD69+. CD4+ T cells were gated by CD4+. CD69+ T cells were gated by CD69+. We performed flow cytometry using a BD FACS Canto™ flow cytometer. FlowJoV10 was used for analyzing flow data.
Statistical analysis
We carried out statistical analysis using GraphPad Prism 8.0. All data on the graphs are presented as mean ± SD. The t-test was adopted for statistical analysis, and a P-value < 0.05 was statistically significant.