Chemicals
Buthionine sulfoximine (BSO), iodoacetic acid (IAA), iodoacetamide (IAM), auranofin (Auro), and carbonyl cyanide m-chlorophenylhydrazone (CCCP) were purchased from Solarbio (Beijing, China); CCCP and Auro were solubilized in dimethyl sulfoxide (DMSO) before use. GX (40%, v/v) was obtained from Xiya Chemical Industry (Jinan, China) and was dissolved in Milli-Q water (Millipore, Burlington, MA, USA) before use. HAECs were purchased from Bena Culture Collection Co. (Beijing, China). Fanconi anemia complementation group A (FANCA) siRNA and siRNA-mate kit were purchased from GenePharma Co., Ltd. (Shanghai, China). The GSH peroxidase assay kit was obtained from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Trx1, phospho extracellular signal regulated kinase (P-ERK), phospho c-Jun N-terminal kinase (P-JNK), and phospho p38 kinase (P-P38) antibodies were purchased from Abcam (Cambridge, UK), and enhanced chemiluminescence (ECL) western blotting substrate was obtained from Biosharp (Hefei, China). The mitochondrial membrane potential kit was purchased from Abbkine (Wuhan, China) and fetal bovine serum was obtained from Tianhang (Zhejiang, China).
siRNA knockdown of FANCA
siRNA-mediated knockdown of FANCA was achieved using the siRNA-mate kit following the manufacturer’s protocol. Briefly, 1 × 105 HAECs were plated with 500 μL of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with fetal bovine serum (FBS) but without antibiotics in each well of a 24 well-plate and incubated at 37 °C in a humidified chamber with a steady supply of 5% CO2. After 24 h of incubation, 30–50% confluent cells were transfected with siRNA targeting FANCA to inhibit its expression. FANCA siRNA (50.1 nmol/L) and siRNA-mate transfection reagent (6 μL) were gently mixed at 20–30 °C for 10 min in 600 μL of DMEM without FBS to form siRNA–liposome complexes, which were then added to each well containing cells. HAECs were incubated in this transfection media for 8 h. The HAECs were continued to be maintained in DMEM with 10% inactivated FBS at 37 °C in a humidified incubator with 5% CO2 for 48–96 h. After incubation, the lysates were harvested for western blot analyses.
Cell culture and survival assays
HAECs were cultured as described previously. For the survival assays with GX, 12 h after plating of the cells, the medium was replaced with fresh medium containing a range of indicated concentrations of GX (0, 50, 100, 150, and 200 μM) every day, and the cells were incubated for 8 days to form colonies. For survival assays, BSO, which can specifically inhibit γ-glutamylcysteine synthetase, was used to deplete the intracellular GSH concentration. HAECs were incubated in culture medium with 1 mM BSO for up to 72 h. Auro, an inhibitor of thioredoxin reductase which specifically interferes with the reduction of oxidized Trx1, was applied at a concentration of 0–6 µM for 24 h, while HAEC membrane permeability to protons was augmented with CCCP treatment (0–800 µM for 2 h).
HAECs were rinsed twice with PBS after treatment; then, the cells were incubated in fresh medium for 8 days to form colonies. Only colonies with more than 50 cells were counted. Drug lethal dose (LD90) values were determined based on the GX dose that produced 90% cell damage, as confirmed from the survival curves.
Measurement of GSH levels
To determine the critical targets of GX, we assessed the changes in the intracellular GSH level. HAECs were treated with GX (LD90). Untreated cells were used as the negative control group. Using a GSH detection kit, GSH was measured immediately after treatment.
The biochemical principle of this protocol is that dithionitrobenzoic acid reacts with sulfhydryl compounds resulting in a yellowish coloration of the compounds, a process which is catalyzed by GSH. Briefly, HAECs (1 × 106) were suspended and lysed by ultrasound after treatment with GX (LD90, 0.12 mM, for 8 days) or BSO. The samples were mixed with GSH (1 mM) and reagent 1 and incubated at 37 °C for 5 min. Following this, a reaction mixture of reagents 2–5 was added to the samples and incubated at 37 °C for 15 min. The GSH levels were measured using an absorbance microplate reader (Molecular Devices, San Jose, CA, USA) at 420 nm.
Measurement of the redox state of Trx1
The samples with reduced and oxidized cysteine thiol groups were treated with GX, and Trx1 proteins bearing different charges were separated by urea-polyacrylamide gel electrophoresis (PAGE) and selectively detected by western blotting [22].
Preparation of electrophoretic migration markers
All cysteine residues were reduced by incubating HAECs with 3.5 mM dithiothreitol (DTT) in urea buffer (8 M urea, 100 mM tris–HCl [pH 8.2], and 1 mM EDTA) for 30 min at 37 °C. Electrophoretic migration markers were treated with 15 mM IAA + 15 mM IAM for alkylation at 37 °C for 15 min.
Preparation of the Trx1 proteins with an unknown redox state
After treatment with GX (LD90, 0.12 mM, for 8 days) or Auro, the Trx1 proteins were alkylated with 200 μL of urea buffer (30 mM IAA) for 15 min at 37 °C. Two milliliters of acetone–1 N HCl (4 °C, 98:2 v/v) was used to precipitate proteins, and unreacted IAA was removed by microcentrifugation for 5 min at 11,000 g at 4 °C. The pellet was resuspended and washed in acetone–1 N HCl–H2O (4 °C, 98:2:10 v/v/v) thrice by microcentrifugation for 5 min at 11,000 g at 4 °C. TRX1 proteins were then reduced by incubating them with 200 μL of urea buffer containing 3.5 mM DTT for 30 min at 37 °C. Trx1 proteins were then mixed with 10 mM IAM for 15 min at 37 °C.
Detection of the redox state of Trx1
The treated Trx1 samples and the electrophoretic migration markers were analyzed with an 8 M urea–PAGE gel, using a 2.5% stacking gel and 12% separating gel run at a constant current of 5 mA for 3.5 h. Proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Darmstadt, Germany) after electrophoresis and incubated at 4 °C for 24 h in TBST (20 mM tris–HCl [pH 7.6], 140 mM NaCl, and 0.1% Tween). The membranes were probed with the relevant primary antibody (1:20,000, ab109385, Abcam) at 4 °C overnight, and rinsed thrice with TBST. They were then incubated with the enzyme-labeled goat anti-rabbit IgG (1:5000) at 37 °C for 2 h. The membrane was incubated with the ECL western blotting substrate after washing with TBST. Chemiluminescence was quantified on a ChemiDoc XRS + system (Bio-Rad, Hercules, CA, USA) and the results were determined using the ImageJ software (Version 1.47).
Measurement of the mitochondrial membrane potential
To determine critical GX targets, we determined the effect of GX on mitochondrial function. HAECs were treated with GX (LD90, 0.12 mM, for 8 days) or without GX (control) and subsequently assessed using a mitochondrial membrane potential assay kit. The measurements were taken immediately after treatment. Briefly, cells were washed and seeded onto a 96-well plate with 100 μL buffer/well. After treatment with GX (LD90) or CCCP, a working solution of the carbocyanine dye JC-1 (100 μL/well) was added, followed by incubation in the dark at 37 °C for 10 min. Results were measured on a Hitachi F-2500 fluorescence spectrophotometer (Tokyo, Japan) at λ ex = 529 nm and λ em = 590 nm.
Western blotting
HAECs were washed with PBS and harvested for treatment with the protein inhibitor (Hefei, China); 30 μg of proteins were subjected to separation on a 10% resolving sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel. The separated proteins were then transferred onto PVDF membranes using a blotting instrument (Bio-Rad). The samples were incubated with the relevant primary antibody at 4 °C overnight, after which they were blocked with 5% bovine serum albumin (20 mM tris–HCl [pH 7.6], 140 mM NaCl, and 0.1% Tween) for 2 h at 37 °C. The steps thereafter were the same as those mentioned in “Detection of the redox state of Trx1” section after incubation with the primary antibody.
Measurement of DPCs
HAECs were treated with GX (LD90, 0.12 mM, for 8 days) or without GX (control) to measure DPCs [23]. Briefly, cells were processed with sarkosyl, mixed with 9.3 g of CsCl (Wako, Japan), and sedimented at 500,000 g at 20 °C for 4 h (CsCl density gradient centrifugation) to isolate chromosomal DNA. Fluorescein isothiocyanate was dissolved in dimethylformamide to a final concentration of 0.1 mM to label DPCs. DPCs were measured with a Hitachi F-2500 fluorescence spectrophotometer (Hitachi, Japan) (λ ex = 490 nm, λ em = 520 nm).
Measurement of DSBs
HAECs were irradiated with X-rays (marker), treated with GX (LD90, 0.12 mM, for 8 days) or without GX (control), after which DSBs [19] were detected by static-field gel electrophoresis. Cells (1 × 104) were loaded on plug molds, and the plugs were slid into 0.6% SeaKem gold agarose gels; electrophoresis was performed for 36 h at a field strength of 0.6 V/cm in 0.5× TBE buffer to isolate DSBs. The samples were detected with a UV transilluminator and the images was captured using a digital camera (Bio-Rad). The band intensity was subsequently determined using the ImageJ software (Version 1.47).
Statistical analysis
Data are presented as mean ± standard deviation of results from three to five repetitions. Statistical analysis was performed using the SPSS 22 package. The t-test was used to evaluate the statistical differences for paired data.