2.4.1 Mammalian cell culture media, antibiotics and supplements.
1) Penicillin and streptomycin solution (Sigma-Aldrich) (100x) 10,000 units penicillin
10 mg/ml streptomycin
2) L-Glutamine solution(Sigma-Aldrich) (100x) 200 mM L-Glutamine
* These two solutions were frozen at –20°C. They were thawed in a water bath at 37°C for at least half an hour prior to use.
3) Fetal calf serum (FCS) (Gibco®, Life Technologies)
This serum was aliquoted and frozen at –20°C. It was thawed at 37°C for at least half an hour prior to use.
4) DMEM medium with high glucose (Gibco®, Life Technologies)
This 500-ml DMEM medium was supplemented with 5 ml of 200 mM L- glutamine (100x), 90 ml of FCS and 5 ml of 100x penicillin and streptomycin solution.
5) Coon’s Modified Ham’s F12 medium (Sigma-Aldrich, F6636)
Nutrient Mixture F-12 Coon's modification powder including 0.292 g/l L- glutamine and 0.863 mg/l zinc sulphate was added to a litre of water. 2.68 g/l sodium bicarbonate (NaHCO3) was added in the solution. This medium was
prepared according to the manufacturer’s instructions (Sigma-Aldrich). Then 500 ml of the medium was supplemented with 100x penicillin and streptomycin solution and 60 ml of FCS.
2.4.2 Cell culture
HeLa, MRC5, MCF7, MDA-MB231 cells were cultured as monolayers in DMEM medium supplemented with 15% fetal calf serum (FCS) and incubated at 37°C with 5% CO2 for 1–3 days to reach 70–80% confluence. PC3 cells were grown as a monolayer in
Coons Modified Ham’s F12 medium containing 2mM glutamine and 12% FCS and incubated at 37°C with 5% CO2 for 1–3 days to reach 70–80% confluence. Details of
these cells are shown in Table 2.3.
Table 2.3 Human cell lines used in this study.
Cell line Organism Origin Cell type Disease
HeLa* human cervix epithelial adenocarcinoma
MRC5 human lung fibroblast fibroblast
MCF7 human breast epithelial adenocarcinoma
MDA-MB231 human breast epithelial adenocarcinoma
2.4.3 Trypsinization of adherent cells
To trypsinize the adherent cells, they needed to be at 70–80% confluence. The medium was aspirated off in a biohazard hood. The cells were washed twice with PBS. 0.25% trypsin solution was added. The flask or plate was incubated at 37°C with 5% CO2
for 5 minutes for trypsin to work. One volume of warm medium was added and the mixture was transferred into a sterile tube. The tube was then centrifuged at 1,500 g for 5 minutes at room temperature. The supernatant was carefully discarded in the hood. The pellet was resuspended in an appropriate medium.
2.4.4 Freezing of mammalian cells
After cells were trypsinized, they were counted in a haemocytometer. They were then pelleted in a conical tube by centrifugation at 1,500 rpm for 5 minutes. The pellet was resuspended in freezing medium (10% DMSO, 90% completed media which containing FSC) to dilute to a final concentration of 1 × 106 cells/ml. 2 ml of the
resuspension was aliquoted in each cryotube vial. The tube was kept in a thermal insulated box and this was placed in a –80°C for 24 hours. Then the vials was stored in a liquid nitrogen container.
2.4.5 Thawing of mammalian cells
A vial was taken from the liquid nitrogen container and thawed rapidly by swirling in a 37°C water bath. The outside of the tube was sterilized with IMS. Cell solution was dropped carefully onto the top of 10 ml of pre-warmed media containing 10% DMSO in a new conical tube. The tube was centrifuged at 1,500 rpm for 5 minutes. The supernatant was aspirated off and the pellet was resuspended in the growth medium. The cells were grown in the same conditions as previously described.
2.4.6 Arsenite treatment
60–80% confluent cells were treated with 1 mM sodium arsenite and then incubated at 37°C for 30 minutes.
2.4.7 Radiation sensitivity test
Cells were irradiated using a [137Cs] gamma source with a dose of 3 Gy, and then
2.4.8 Immunofluorescence
1 × 105 mammalian cells were cultured in 2 ml of the appropriate media in a six-
well plate containing cover slips in each well at 37°C for 48 hours. The cells needed to be at 60–80% confluent for treatment with any conditions. The supernatant was aspirated off and the cells were washed twice with PBS. The cells were fixed and permeabilized with cold absolute methanol for 20 minutes at –20°C. The cells were blocked with 2% BSA (bovine serum albumin) in PBS for 30 minutes at room temperature. The BSA solution was removed and replaced with appropriate dilution of primary antibody in 2% BSA. The primary antibodies were typically used at a 1:50 dilution in 2% BSA. From this step on, the cells were kept in a humid atmosphere to prevent them from drying out. The reaction was incubated for 1 hour at room temperature after that the cells were washed three times with PBS. The 1:200 dilution of specific secondary antibody in 2% BSA was then added. The reaction was incubated for at least 40 minutes at room temperature. The cells were washed three times with PBS. To mount the cells, 15 µl of ProLong® Gold Antifade Reagent with DAPI was dropped on a slide and the coverslip containing the cells was mounted upside down and placed on top of the DAPI reagent. The reaction was incubated for an hour at room temperature. Slides were kept at 4°C prior to examination by wide field fluorescence microscopy (DeltaVision) according to the manufacturer’s instructions.
Table 2.4 List of primary and secondary antibodies used in immunofluorescence assay
Antibody Source Type Clone Company Dilution
anti-eIF4G mouse primary monoclonal Santa Cruz (sc-373892)
1:50
anti-eIF4E rabbit primary polyclonal In-house 1:100 anti-eIF4A rabbit primary polyclonal In-house 1:100 anti-SUMO1 rabbit primary polyclonal Santa Cruz
(sc-9060)
1:50
anti-SUMO1 mouse primary monoclonal Santa Cruz (sc-5308)
1:50
FITC-anti- mouse IgG
goat secondary polyclonal Sigma (F0257) 1:200
Cy3-anti-rabbit IgG
sheep secondary polyclonal Sigma (C2306)
1:200
2.4.9 Two-step transfection of siRNA and plasmids
A mixture of siRNA was prepared in a 1.5-ml tube by the addition of 50 μl Opti- MEM (31985-047, Life Technologies), 5 μl HiPerFect transfection reagent (301705, QIAGEN) and 10–30 nM siRNA eIF4AII (s4572, Life Technologies). The siRNA mixture was incubated at room temperature for 10 minutes. 2 × 105 HeLa cells were
added in 2 ml DMEM in a six-well plate, followed by the addition of the siRNA mixture, then incubated at 37°C for 48–72 hours. The cells needed to be at approximately 70% confluent. The cells were washed twice by PBS, and 2 ml fresh medium was added into each well. A mixture of plasmid was prepared by the addition of 130 μl Opti-MEM, 6 μl FuGENE® HD transfection reagent (e2311, Promega) and 2–3 μg plasmid DNA, then incubated at room temperature for 10 minutes. The plasmid mixture was added into the six-well plate. This was incubated at 37°C for 24 hours. The cells were harvested for further analysis.