Reagents and chemicals
ON-TARGETplus Non-targeting pool siRNA, TRX (TRX-1) ON-TARGETplus SMARTpool siRNA, TRX-2 ON-TARGETplus SMARTpool siRNA, and Dharmafect I transfection reagent were from Dharmacon Research (Lafayette, CO, USA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), glutathione ethyl ester (GSSE), cycloheximide, and L-Buthionine-[R,S]-sulfoximine (BSO) were purchased from Sigma (St. Louis, MO, USA). Murine TNFα was from Fitzgerald (Concord, MA, USA). Antibody against TRX-1, p-ASK1 and PDI was from Cell Signaling Technology (Beverly, MA, USA). Antibody against TRX-2 was from Santa Cruz Biotechnology INC (Santa Cruz, CA, USA). Antibody against LC3 was purchased from Thermo Scientific (Rockford, IL, USA). Antibody against CYP2E1 was a generous gift from Dr. Jerry Lasker. Fluorescence-conjugated secondary antibodies used in the Odyssey infrared imaging system were from Li-Cor Biosciences (Lincoln, NE, USA). All other antibodies were from Santa Cruz Biotechnology INC (Santa Cruz, CA, USA). Total ROS detection kit was purchased from ENZO Life Sciences (Plymouth Meeting, PA, USA). Histostain Plus Broad Spectrum (ACE) kit was from Invitrogen (Camarillo, CA, USA). The JNK inhibitor, L-JNKI1 was purchased from EMD Chemicals (San Diego, CA, USA). Dihydroethidine (DHE) was purchased from Invitrogen (Camarillo, CA, USA). MitoSOX Red reagent was purchased from Molecular Probes (Eugene, OR, USA). ApoAlert Annexin V was from Clontech (Mountian View, CA, USA).
Cell culture and treatments
C34 and E47 HepG2 cells were cultured either on 96-well plates or 6-well plates or on glass cover slides according to the requirements of the experiments. E47 cells are HepG2 cells which were transfected with PCi plasmid containing human CYP2E1 cDNA. These cells constitutively express CYP2E1. C34 cells are HepG2 cells transfected with the empty plasmid. These cells express low or zero CYP2E1. E47 and C34 cells were maintained in DMEM containing 10% FBS plus 1% penicillin-streptomycin-glutamine mixture plus 0.1 mg/ml G418 [19
]. Cells were plated at a density of 1×104
cells/ml in DMEM containing 10% FBS and 1% antibiotics (penicillin plus streptomycin). SiRNA of TRX-1, TRX-2, TRX-1 and TRX-2 together, or control were used at 10nM concentration in DMEM containing 2% FBS and were performed 24 hrs after cells were plated. The treatment was continued for 5, 24, 48, and 72 hrs. The respective siRNA was present during the entire culture period. The transfection reagent was Dharmafect I used at a concentration of 1µl/ml. For cells treated with either BSO or TNFα, 200µM BSO or 2ng/ml TNFα with 10µg/ml cycloheximide were added after 24 hrs of siRNA treatment. The cells continued to be incubated with the appropriate siRNA and either BSO or TNFα for another 48 hrs (total siRNA treatment for 72 hrs). To test the protection effect of glutathione ethyl ester, 5mM glutathione ethyl ester was added to the E47 cell culture medium after 24 hrs siRNA treatment and the cells were incubated with the respective siRNA and glutathione ethyl ester for another 48 hrs. To test the effect of a JNK inhibitor on cell viability, E47 cells were treated with 5µM L-JNKI1 for 3 hrs, then cells were treated with either control siRNA, or the respective thioredoxin siRNAs for another 72 hrs. Cells were collected and various analyses were carried out as described below.
Cell viability assay
Cells grown on 96-well plates and treated with siRNA for 72 hrs were incubated with MTT for 3 hrs. Cell culture medium was aspirated, and isopropanol was added and plates were shaken for 30 min. Absorbance at 590nm and 630nm was detected on a plate reader. Cell viability was calculated as the absorbance difference between 590nm and 630nm. Cell viability in the control siRNA group of both C34 and E47 cells was taken as 100%, and cell viability in the thioredoxin knockdown groups was expressed as the percentage of viability relative to that of the control siRNA group in corresponding C34 and E47 cells.
Propidium iodide and Annexin V staining
Cells on 6-well plates were treated with siRNA for 72 hrs. For PI staining, 5µg/ml PI was added to the culture medium and the cells incubated for 10 min at 37 °C. PI staining was analyzed using fluorescence microscopy. Images shown were the merged ones taken with both light and fluorescence microscopy. The ApoAlert Annexin V detection kit was used for Annexin V staining. After siRNA treatment for 72 hours, cells were digested with trypsin, and pelleted by centrifugation. Cells were washed twice with binding buffer supplied in the detection kit, then resuspended with 200µl binding buffer, treated with 5µl Annexin V stock solution (20µg/ml) and incubated for 15 min in the dark. Annexin V staining was analyzed by flow cytometry in the FITC channel.
ROS detection by microscopy
ROS was detected with the Total ROS Detection Kit from Enzo. Cytosolic ROS was detected by fluorescence of DHE, and mitochondrial ROS was detected by fluorescence of mitoSOX Red. Briefly, cells were allowed to attach to the glass cover slides for 24 hrs. Cells were treated with either TRX-1 siRNA, TRX-2 siRNA or both for 72 hrs. Cells were loaded with ROS detection solution or 40 µM DHE or 5 µM mitoSOX Red reagent at 37°C and incubated in the dark. Cover slides were washed with wash buffer twice, then overlaid on slides and observed immediately under a fluorescence microscope. Images were taken at 40× magnitude. Images for mitoSOX were merged images from light microscopy and fluorescence microscopy.
Flow cytometry and spectrofluorometry assay for ROS
Cells grown on 6-well plates were treated with siRNA as described above for ROS detection by microscopy. Cells were harvested by digestion with trypsin. The cells were washed twice with wash buffer. Cells were treated with ROS detection solution for 30 min at 37°C in the dark. Samples were directly analyzed by flow cytometry using the FITC channel or spectrofluorometry (Ex/Em: 490/525nm).
Immunostaining for TRX-1, TRX-2, CYP2E1, SODs, pASK-1, ASK-1, pJNK, JNK, pp38 MAPK, p38 MAPK, p-cJUN, and cJUN
C34 and E47 cells, either treated with control siRNA or thioredoxin siRNAs, were collected typically after 72 hrs siRNA treatment. For the MAPK experiments, cells were collected after 5, 24, 48, and 72 hrs siRNA treatment for staining of pASK-1, ASK-1, pJNK, JNK, pp38 MAPK, and p38 MAPK. Cells were lysed with RIPA lysis buffer containing protease inhibitors. Western blotting was performed with a Bio-Rad mini gel system. Membranes were blotted with corresponding antibody separately for overnight at 4°C. Fluorescence conjugated secondary antibody against mice or rabbit IgG were incubated with the membranes for 1 hr at room temperature in the dark. Fluorescence was detected by an Odyssey infrared imaging system from Li-Cor Biosciences. Quantitation of fluorescence intensity was performed with an Image J program (NIH).
Immunostaining for pASK-1
Cells grown on glass cover slides after 5, 24, 48, 72 hrs siRNA treatment were fixed in 10% buffered formalin for 15 min. Slides were rinsed with PBS twice, and treated with 0.1% Triton X-100 for 15 min. After rinsing twice in PBS, fixed cells were blotted with blotting buffer for 30 min at room temperature. Antibody against pASK-1 was added at 1:100 dilution and incubated at 4°C overnight. Slides were rinsed 3 times with PBS and incubated with Alexa 488 conjugated secondary antibody for 1 hr at room temperature. Fluorescence was detected under a fluorescence microscope at 40× magnitude.
Immunostaining for 4-HNE adducts
Cells grown on glass cover slides after 72 hrs siRNA treatment were fixed in 10% buffered formalin for 15 min. Slides were rinsed with PBS twice, and treated with 0.1% Triton X-100 for 15 min. After rinsing in PBS twice, fixed cells were blotted with blotting buffer for 30 min at room temperature. Antibody against 4-HNE was added at 1:200 dilution and incubated at 4°C overnight. Slides were rinsed 3 times with PBS and then were stained using a Histostain Plus Broad Spectrum (ACE) kit. 4-HNE staining was detected under a light microscope at 20× magnitude.
Total glutathione analysis
Cells were collected after 72 hrs siRNA treatment, and lysed. Cell lysates were mixed with 10% trichloroacetic acid (TCA) and incubated for 10 min to extract intracellular glutathione. The TCA supernatant was used to measure the glutathione content following the method of Tietze [20
]. 0–100µM glutathione dissolved in 5% TCA solution was used for standard curve analysis. Results were expressed as nanograms of glutathione per milligram of protein. The relative GSH level was expressed with the control siRNA treatment group of E47 cells taken as 1.0. The relative GSH level in other groups was expressed as the ratio to that of control siRNA group of E47 cells.
One-Way ANOVA was performed using the Statistical software SPSS (17.0) to compare the difference between control and treatment groups. Values reflect means ± standard error from 3–4 experiments. Statistical significance was expressed as p<0.05.