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HeLa Human epithelial cell line originating from a cervical carcinoma. Adherent cells. HeLA S3 Derived from HeLa. Cells have been adapted to grow in suspension culture Jurkat Human T lymphoblastoid cell line

293T Adenovirus 5-transformed human embryonic kidney cell line. F99 Human skin primary fibroblast

2.2.2. Culture conditions

The cell lines used in this study were grown in the following media:

HeLa, 293T, F99 - Dulbeccoʼs modified Eagle medium (DMEM plus 4500 mg/ml glucose, L-Glutamine, without Pyruvate. Gibco Invitrogen, Cat. No.11971-025) supplemented with 10% heat-inactivated (56 °C for 30 min in a water bath) foetal bovine (calf) serum (FBS=FCS, Gibco Invitrogen, Cat. No.10270-106), 100 units/ml penicillin G and 100 μg/ml streptomycin sulfate (Gibco Invitrogen, Cat. No.15140-

122).

Jurkat and HeLA S3 - RPMI 1640 medium (plus L-Glutamine, Gibco Invitrogen, Cat. No.21875-034), supplemented with 10% heat-inactivated FBS and 100 U/ml penicillin G, 100 µg/ml streptomycin sulfate.

The adherent cell lines, used in this study were usually grown in 10-cm or 15- cm tissue culture dishes in a tissue culture incubator with 5% CO₂ atmosphere at 37 °C. Subculturing of the adherent cells was done by trypsinization with 0.25% trypsin, 0.2% EDTA (Gibco Invitrogen, Cat. No.25050-014) every second day. Cells were usually kept in culture for no more than 20 passages.

Suspension cell lines were grown in 25, 75 or 175 cm2 tissue culture flasks. Subculturing of suspension cells was done by diluting with fresh medium. Cells were kept at concentration between 0.2-1x106 _cells/ml.

Freezing of cells was done in 90% FCS and 10% (v/v) dimethyl-sulfoxide (DMSO) in a freezing box at -80°C. The frozen aliquots were stored in a liquid nitrogen tank or at -80°C.

Thawing of the cells was done by submerging the frozen vial in a 37 °C water bath. The cells were then washed with medium by centrifugation at 1200 rpm for 3 min and resuspended in pre-warmed medium before transferring them to culture plates or flasks.

2.2.3. Cell syncronization

Unless specified, adherent HeLa cells were used in the following synchronization protocols. Cells were maintained exponentially growing, splitting them regularly every second day. To increase the efficiency of synchronization, cells were always plated the day before to obtain dishes ca. 60% confluent when exposed to the specific inhibitors.

100 Materials and Methods

2.2.3.1. Mitotic block

At mitosis (M) most vertebrate cells round-up. In many cell lines, especially those of epithelial origin, which grow in monolayer, attachment to the substrate also becomes much loser. This causes some mitotic cells to detach spontaneously. The mitotic arrest can be induced chemically using nocodazole, an inhibitor of microtubule polymerization. An almost pure population of M-blocked cells can be obtained combining the nocodazole block to mechanical shaking. Cells were treated with 600 ng/ml nocodazole (Sigma) for 16 h before harvesting.

2.2.3.2. Mitotic block and release into G1

Cells were exposed to 50 ng/ml nocodazole for 17 h, the mitotic cells were collected by gentle pipetting, and washed in DMEM at 37 °C, resuspended in DMEM/10% FCS, replated and cultured for an additional 6 h. The mitotic cells obtained from four 15 cm plates were plated onto one 10 cm plate.

2.2.3.3. Late G1 block

L-mimosine is a plant aminoacid that provokes synchronization of cells just before the start of the S phase. HeLa cells were arrested in late G1 exposing them to 400 μM mimosine (Sigma) for 20 h before harvesting.

2.2.3.4. S and G2 block

Any inhibitor of DNA synthesis arrests replicating cells. The most used are inhibitors of the synthesis of deoxyribonucleotide triphosphates, as thymidine at high concentrations. A double-arrest accumulates more than 90% of the population. HeLa cells were exposed to 2 mM thymidine for 17 h, rinsed twice, released in growth medium for 9 h and then blocked again with 2 mM thymidine for 15 h. At this point cells are synchronized at late G1. To obtain a G2 population, cells were washed and let grow in normal medium for 8 h.

2.2.4. Metabolic labeling of mitotic and asynchronous cells

At 13 h after the addition of nocodazole, the mitotic detached cells of four 15 cm plates were collected, washed twice and cultured in a well of a 6-wells plate with 7 ml of H₃PO₄-free medium (Dulbeccoʼs Modified Eagle Medium, with high glucose and L-Glutamine, without Sodium Phosphate and Sodium Pyruvate Gibco Invitrogen, Cat. No. 11971-025), supplemented with 20 mM Hepes _{buffer, penicillin-}

streptomycin, 10% Fetal Bovine Serum (FBS) Dialyzed (H₃PO₄-free. Gibco, Cat. No. 26400-036), and 600 ng/ml nocodazole. After 2 h cells were centrifugated and

resuspended in 2 ml of the same medium supplemented with [32_P]H

3PO4 (0.5 mCi / 1 ml of medium) (NEN Life Science Products, Perkin Elmer, Cat. No. NEX053). Cells were labeled for 5 h before harvesting.

The sample of exponentially growing cells was exposed to H₃PO₄-free medium containing 600 ng/ml nocodazole and 10% FBS (H₃PO₄-free) for 2 h, and then 5 h more with [32_P]H

3PO4. One 12 cm plate was used for the exponentially cells and the plate was covered with 2.5 ml medium (0.5 mCi / 1 ml of medium).

In order to preserve protein phosphorylations, the cell lysis buffer (see below) contained in addition 1μM okadaic acid.

2.2.5. Calcium phosphate transfection

Transfection was performed according to the protocol described in Chen and Okayama (1988).

The day before transfection, 293T or HeLa cells were seeded in 6-well plates at 30% confluency. 20 μg of total DNA (the concentration in each transfection experiment

was kept constant by adding vector plasmid DNA) were added to 500 μl of 250 mM

CaCl₂, and then the tubes were vortexed. 500 μl of 2xHBS were added dropwise

while gently vortexing, and the solution was incubated 20 min at RT. The calcium phosphate-DNA solution (160 μl / well) was added dropwise onto the cell culture

plate while swirling. The plates were incubated overnight in a 5% CO₂-humidified incubator at 37 °C to allow for a calcium phosphate-DNA complex to gradually form. The cells were then washed three times in PBS, and incubated in complete medium in a 5% CO₂ humidified incubator at 37 °C.

2.2.6. Polyfect transfection

Transfection of HeLa cells with PolyFect transfection reagent (Quiagen, Hilden) was conducted following the manufacturer. For each well of a 6-well plate, 1.5 μg of

DNA dissolved in TE buffer was diluted in 100 μl OPTIMEM 1 (with Glutamax, Gibco

Invitrogen, Cat. No. 51985-026) before adding 12 μl of PolyFect transfection reagent

was added. Note that this reagent is toxic for the cells, and the day after transfection dead cells were observed in the supernatant, also in the control (cell transfected with dH₂O). Therefore cells were always fixed for the immunofluorescence 16 to 24 h after transfection.

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