3.1.1 Cell Culture
For most cell based experiments described in papers I-VI, commercially available human cell lines from the American Type Culture Collection were used. Fibroblast cells in paper I was a gift from Dr. Laura Papp, Queensland Institute of Medical Research, Australian. Hela cell (human cervical carcinoma), A549 (human alveolar adenocarcinoma epithelial cell), SH-SH5Y (human neuroblastoma) and Du145 (human prostate cancer) were cultured in Dulbecco's modified Eagle's medium (DMEM) with 1 g/l glucose (VWR). HEK293t (human embryonic kidney cell) and Fibroblasts were cultured in RPMI 1640 medium with 1 g/l glucose (VWR).
3.1.2 RNA Silencing
The transfection of siRNA in Paper I and Paper III was performed according to the Dharmacon transfection protocol. Briefly, siRNA transfection reagent (Dharmacon, Lafayette, Co, USA) and siRNAs (Qiagen, Valencia, CA, US) were diluted in serum and antibiotic-free medium and left at room temperature for 5min. Then the siRNA transfection reagent and siRNA were mixed and incubated for 20 min. Complete medium were added into the mixture to achieve a final concentration of 50 nM siRNA in the culture medium. After 48 hours incubation, the medium was removed, and the cells were harvest for analysis or continue incubated with the desired compounds.
3.1.3 Cell Proliferation and Viability Assays
The most commonly used assay for cell proliferation and viability measurement in all six papers is the MTT assay. Because MTT assay can be affected by the alteration of mitochondrial metabolism, in order to confirm the results of MTT assay, we also used trypan blue exclusion and neutral red up take assay to investigate the effects of compounds we used on cell viability in some of our studies. Observations of the
morphology of cells by microscopy were always performed and recorded prior to each assay.
For MTT assay, cells were plated at a density of 1×104 cells/well in 96-microwell plate and allowed to grow about 24 hours to get confluence. Then the cells were treated with appropriate concentrations of compounds in 100 µl fresh medium for the appropriate time. After treatment, medium containing compounds were replaced by 100 µl of fresh medium, and 50 µl of MTT solution (1 mg/ml in PBS) was added to each well and incubated for 3h. Then the medium was carefully removed, and 100 µl of DMSO was added to each well. Plates were then put on a shaker for about 1 hour until all crystals were dissolved. Then the cell viabilities were determined by measuring the absorbance at 550 nm.
Neutral red up take assay was carried out following the previous described protocol168; cells were seeded and treated in the same way as in MTT assay. Neutral red working solution (40 µg/ml in culture medium) was incubated overnight at the same temperature as the cells, and then centrifuged to remove any precipitated dye crystals. The treatment medium was then removed and 100 µl of neutral red medium were added into each well. The plate was incubated for 2 hours at the appropriate culture conditions. Then the neutral red medium was removed and the cells were washed with 150 µl PBS carefully. Then 150 µl neutral red destaining solution (50% ethanol 96%, 49%
deionized water, 1% glacial acetic acid) was added into each well. The plate was then placed on a plate shaker until the neural red has been extracted from the cells. Then the cell viabilities were determined by measuring the absorbance at 540 nm.
For trypan blue exclusion assay, after the treated cells were collected, cells were centrifuged and resuspended in PBS. The density of the cells was determined using a hemocytometer. Then in every 1 ml cell suspension, 0.1 ml of trypan blue stock solution (0.4% trypan blue in PBS) was added. The numbers of the blue staining cells were counted right away, and the cells which took up trypan blue were considered non-viable.
3.1.4 Measuring TrxR activity using fluorescent method
Insulin reduction assay and DTNB reduction assay are two most commonly used method to measure TrxR activity in purified protein or in cell lysates. But in order to detect TrxR activity in precious biological materials, such as cell lysates from different subcellular organelles, or cell samples containing a low amount of TrxR, a newly developed fluorescent method was applied in the studies169. In the fluorescent method, insulin was replaced by isothiocyanate-labeled insulin (FiTC-insulin), which emits fluorescence at 520 nm after excitation at 480 nm. Upon the reduction of the disulfide by Trx, the fluorescence is increased. Compared to the conventional methods, fluorescence method is highly sensitive and stable169.
Generally, in a 96-well black micro titer plates, appropriate amount of cell lysates were incubated with 20 µM of Trx and 0.25 mM NADPH in assay buffer (0.2mg/ml bovine serum albumin in 50 mM Tris-Cl and 1 mM EDTA, pH 7.5), the total volume was 90 µl in each well. After incubation 30 min at 37°C, 10 µl of FiTC-insulin was added into each well. The final concentration of FiTC-insulin in each well is 10 µM. Then the emission at 520 nm after 480 nm excitation was recorded for 60 min in room temperature. The rate of the reaction was calculated as the changes of fluorescence following time. In paper IV, due to the very low amount of TrxR in SH-SH5Y cells, an improved fluorescent method was used, in which FiTC-insulin was replaced by a new fluorescent substrate from the latest developed kit (FkTRXR-03-Star) by IMCO (www.imcocorp.se).
3.1.5 Redox Western Blot
To determine the redox state of Trx1 in cells, the experiment was performed based on the method described previously38,166. Cells were washed three times in cold PBS and then lysed in urea lysis buffer containing iodoacetamide (IAM) (10 mM IAM, 50 mM Tris-HCL, 1mM EDTA, 8 M urea, and pH 8.3). Then the proteins were precipitated and washed three times to remove the excess IAM with 1.5 ml of ice-cold acetone/HCl (98/2, v/v). The precipitate was then resuspended in urea lysis buffer containing 3.5 mM DTT to reduce the disulfides in Trx1. The free thiols of Trx were then alkylated with 30 mM iodoacetic acid (IAA) in urea lysis buffer. The samples were then
separated by PAGE containing 8 M urea and transferred with an Invitrogen transfer system.
Figure 4. Principle of modified redox western blot (from paper II )
As exhibited in Fig. 4, in 8 M urea, proteins were all denatured and all the thiols were exposed. When first incubated with IAM, the free thiols in Trx1 were labeled with IAM, which will not give any extra charge to the protein. After removing excess IAM, the thiols which are oxidized into disulfide or bind to other proteins were reduced by 3.5 mM DTT, and were labeled by IAA. IAA is negatively charged, so the protein labeled with more IAA will migrate faster in the urea gel. In the end, Trx1 can be separated according to the free thiols they contained initially.