1. Cell Culture and Treatment
Human lens epithelial cells HLE-B3 (also named LECs), immortalized by infection with adenovirus 12-SV40 (ATCC, USA), were grown and maintained in 1:1 Dulbecco’s modified Eagle’s medium and Ham’s F-12 medium (DMEM/F-12) (#10-092-CV, Corning Cellgro, USA) which is supplemented with 10% fetal bovine serum (FBS) (Bioind, Israel), 100 U each of penicillin and streptomycin (Gibco, USA) in humidified CO2 incubator. For overexpression of Trx-1, the LECs were transfected with Lipofectamine 2000 and vector-control (vector) or TBP-2-overexpression lentivirus (TBP-2), which were designed and constructed by Shanghai Genechem.
Cells were plated 24 hours before the experiments and experiments were performed when cell confluence reached 80%-90%. Before the treatment, cells were washed with PBS twice to avoid the influence of serum. Cells were treated with 50 μM H2O2 in DMEM/F12 without serum for different durations. According to our previous research[11], in wortmannin and bafilomycin A1 inhibitory experiments, cells were treated with complete medium or 50 μM of H2O2 was treated for 12 hours. In these 12 hours, 10 𝜇M wortmannin (#S2758, Selleck, USA) was added to the medium after six hours; 50nM bafilomycin A1 (#BF1116, Sangon, China) was added at the ninth hour.
2. Real-time reverse transcription-polymerase chain reaction
Total RNAs were isolated from cells using Trizol (#15596026, Invitrogen, USA). The reverse transcription (RT) was conducted by the PrimeScript™ RT Master Mix (#RR036A, Takara, Japan), and the products were subjected to real-time polymerase chain reaction (PCR) analysis using a TB Green™ Premix Ex Taq™ II (#RR420A, Takara, Japan) with an Applied Biosystems7500 Fast Real-Time PCR System (Applied Biosystems). PCR primers specific for Trx-1, Trx-2, TBP-2 and beta-actin. were designed with the help of website - Primer bank[12] (Table 1). Dissociation curves of PCR products were obtained to guarantee amplification of the correct genes. Each sample was run in triplicate for technical repeats. The mean threshold cycle value of the triplicates and the n-fold difference in expression of the genes between different groups were determined after normalization to the expression level of beta-actin.
Table 1: The oligonucleotides served as the primers of qRT-PCR.
Gene symbols
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Primers
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beta-actin
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Forward
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5’-CATGTACGTTGCTATCCAGGC-3’
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Reverse
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5’-CTCCTTAATGTCACGCACGAT-3’
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Trx-1
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Forward
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5’-GTGAAGCAGATCGAGAGCAAG-3’
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Reverse
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5’-CGTGGCTGAGAAGTCAACTACTA-3’
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Trx-2
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Forward
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5’-CTGGTGGCCTGACTGTAACAC-3’
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Reverse
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5’-TGACCACTCGGTCTTGAAAGT-3’
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TBP-2
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Forward
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5’-AAGCTGTCCTCAGTCAGAGGCAAT-3’
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Reverse
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5’-ATGACTTTCTTGGAGCCAGGGACA-3’
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3. Protein extraction and Western Blot analysis
The mitochondrial fraction was isolated according to the protocol of Mitochondria Isolation Kit for Cultured Cells (#89874, Thermo, USA). Briefly, the cell pellets were vortexed in 800μl of buffer A at medium speed for 5 seconds, and incubated on ice for exactly 2 minutes. Then tube was added in 10μl buffer B to vortex at maximum speed for 5 seconds. After that, tube was incubated on ice for 5 minutes, vortexing at maximum speed every minute. 800μl buffer C was added in tube to mix, followed by centrifugation at 700 g for 10 minutes at 4°C to collect nuclei and membranes. The chilled supernatant after centrifugation was considered the cytosolic fraction and the sediment was the mitochondrial fraction. The supernatant was centrifuged at 12,000 g for 15 minutes at 4°C. The supernatant was cytosol fraction, the pellet contained the isolated mitochondria. The sediment was resuspended in 500 μL of buffer C and centrifuged again at 12,000g for 5 minutes. The final sediment was lysed with 2% CHAPS in Tris-buffered saline. The subcellular fractions were validated by Western blot using cytosolic and mitochondria markers (See Supplementary Figure S1, Additional File 1).
The BCA Protein Assay Kit (#23225, Thermo, USA) was used to determine the protein concentration. Same amounts of protein were subjected to SDS-PAGE, transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, USA) and blocked for 1 hour with 5% nonfat dry milk in Tris buffered saline (TBS-T, 10mM Tris-HCl [pH 7.5], 100mM NaCl, and 0.1% Tween 20). Primary antibody incubations were performed at the following dilutions: Trx-1, 1: 2000 (#ab86255, Abcam, USA); Trx-2, 1: 100 (#sc-133200, Santa Cruz Biotech, USA); Voltage-dependent Anion Channel (VDAC), 1: 5000 (#ab15895, Abcam, USA); beta-tubulin, 1:3000 (#EM1701-59, Huaan Biotechnology, China); TBP-2, 1: 200 (#K0204-3, MBL, Japan); Microtubule-associated protein 1A/1B-light chain 3 (LC3), 1: 1000 (#L7543, Sigma-Aldrich, USA); p62, 1: 1000 (#8025, CST, USA); Glyceraldehyde 3-phosphate Dehydrogenase (GAPDH), 1: 5000 (#M20006, Abmart, China). All incubations were done at 4°C overnight. Then, the membrane was washed thrice with TBS-T for 30 minutes and followed by respective secondary antibody incubations with goat anti-rabbit/mouse IgG-horseradish peroxidase (1:5000, Santa Cruz, USA). Immunodetection was performed with the ECL Western Blot Detection System (Millipore, USA). The immunoblot was analyzed with an imaging system (ChemiDoc™ MP System, Bio-Rad, USA). The intensities of the bands were quantified by densitometry using Image Lab (Bio-Rad, USA). Values were reported as percentage of the control values ± SEM.
4. Trx-1 activity evaluation
Trx-1 activity was evaluated by Thioredoxin Activity Fluorescent Assay Kit (#11527, Cayman, USA). Briefly, Cell lysates was added in a certain volume of assay buffer, TrxR, beta-NAPDH and fluorescent substrate in sequence, followed by recording the emission at 545 nm after 520 nm excitation for 60 minutes in a fluorescent plate reader at room temperature. The obtained data was calculated according to the increasing fluorescence intensity for the time of the reaction within a liner range.
5. Immunofluorescence staining(IF)
Cells were seeded on cleaned coverslips 24 hours prior to the treatment. After H2O2 treatment, the cells were fixed in 4% formaldehyde solution. Next, the cells were permeabilized with 0.2% Triton-X-100 (Sigma-Aldrich, USA) and blocked with 20% goat serum (Applygen, China). Subsequently, the cells were incubated with anti-Trx-1 (#ab86255, Abcam, USA) or with anti-Trx-2 (#ab185544, Abcam, USA) followed by anti-TBP-2 (#K0204-3, MBL, Japan) overnight at 4°C. After thorough washing with PBS, Alexa Fluor® 488 Goat Anti-Rabbit IgG (#150077, Abcam, USA), Alexa Fluor® 488 Goat Anti-Mouse IgG (#150113, Abcam, USA) or Alexa Fluor® 555 Goat Anti-Mouse IgG (#ab150114, Abcam, USA) solution was used to incubate the cells for 1.5 hours at room temperature. The mitochondria were stained with MitoTracker® Deep Red FM (#M22426, Thermo, USA). The nuclei were stained with DAPI. Eventually, the coverslips were mounted on the slides with a drop of mounting medium (H-1000, Vector, USA). Fluorescent images were captured with a Leica DM2500 Fluorescence microscope.
6. Cytotoxicity Assay
Cytotoxicity assays were performed using a Cell Counting Kit-8 (CCK-8) assay (Dojindo, Japan). The cells were incubated with indicated concentrations of H2O2 dissolved in DMEM/F12 without serum for 12 or 24 hours. After the treatment, the medium was removed and the cells were washed with PBS. Then, each well was refilled with 90 μL of DMEM/F12 supplemented with 10% fetal bovine serum and 10 μL of CCK-8 reagents and incubated in 37°C for 3 hours. The cell viability was evaluated with OD450 values using a 96-well microplate reader (Bio-Rad, USA).
7. Co-Immunoprecipitation
Anti-TBP-2 antibody was used for the immunoprecipitation of TBP-2 and Trx-1 complex. The cytoplasm proteins were incubated with 2μg anti-TBP-2 (#K0204-3, MBL, Japan) overnight at 4 °C. Then the immunoprecipitated complexes was incubated with magnetic beads (Bio-Rad, USA) for 4 hours at 4 °C, washed three times with ice-cold washing buffer, resuspended in electrophoresis SDS sample buffer, boiled for 5 min at 95 °C, and analyzed on 10% SDS-PAGE. Immunoblotting was performed using the following primary antibodies: anti-TBP-2 (1:2000, #ET1705-72, Huaan Biotechnology, China), anti-Trx1 (1:2000, for cytosol fraction; #ab86255, Abcam, USA). The secondary antibodies used were as follows: goat anti-rabbit/mouse IgG-horseradish peroxidase (1:5000, Santa Cruz, USA)
8. Statistics
Three independent replications were performed, and the numerical data were presented as mean ± standard error of mean (SEM). The differences between control and experimental groups were tested for significance with the independent Student’s t-test or one-way ANOVA. The independent Student’s t-test was used for comparison between the two groups, one-way ANOVA for multiple comparisons between the groups. A difference of p<0.05 was considered statistically significant.