Buffers and Solutions

All solutions w ere prepared using distilled-deionised w ater and w ere stored at room tem p eratu re (20°C) unless otherw ise stated. Solutions are listed overleaf, alphabetically.

SOLUTION COMPONENTS

A crylam ide Aery lam ide / Bisacry lam ide solution (37.5:1). M ade from p re­ w eighed pow der (BioRad) by adding ddH20 and stirring for 30 m inutes at room tem perature. Stored at 4°C in foil w rapped bottles.

A m m onium p ersulphate (APS) 10% (w/v) APS (BioRad) freshly prepared on day of use.

Buffer P 2mM MgCl2, Im M ATP, Im M

EGTA, 12mM K Phosphate buffer, pH 6.8.

Coom assie Blue Stain 50% (v /v ) m ethanol (BDH), 10% (v /v ) glacial acetic acid (BDH), 0.05% (w /v ) coomassie Brilliant Blue R (Sigma).

D estain 30% (v /v ) m ethanol (BDH), 10% (v /v ) glacial acetic acid (BDH).

Dulbecco's Modified Eagle's M edium (DMEM)

Commercially purchased and stored at 4°C.

Electroblot Transfer Buffer

25mM Tris base (Sigma), 192mM glycine (Sigma), 20% (v /v )

m ethanol (BDH); pH~8.3.

Laemm li Sam ple Buffer 80mM Tris-Cl p H 6.8, 2.3% SDS

(Pierce), 0.002% brom ophenol blue (BDH), 1.42M 2-m ercaptoethanol (BDH), 20% glycerol (Fluka)

(Laemmli, 1970). M ade fresh on day of use.

Laem m li R unning Buffer Ix 25mM Tris Base, 192mM glycine (Sigma), 0.1% SDS (Sigma), pH~8.3.

2-m ercaptoethanol 14.2M 2-ME (BDH). Stored at 4°C in

dark bottles.

Phosphate Buffered Saline IxPBS. 137mM NaCl (Sigma), 2.7mM KCl (Sigma), 8mM

N aH2P0 4 (Sigma), 1.45mM

KH2PO4 (Sigma); pH>7.5.

2 . 2 Tissue C ulture

2.2.1 M edia an d Plastics: All m edia used in these experim ents w ere supplied by Sigma and stored at 4°C. Unless otherw ise indicated, m edia w ere

su p p lem en ted w ith 10% foetal calf serum (ECS) (Sigma) w hich h ad been p rev io u sly h eat treated at 56°C for 30 m inutes to inactivate com plem ent, aliquoted and stored at -20 °C. Also ad d ed to DMEM w ere 4000 u n its /litre

p en icillin (Sigm a), 4000|Xg/litre stre p to m y cin (Sigma) an d 5 8 4 m g /litre glutam ine (Sigma).

Cells w ere cultured in 25cm^, 75cm^, or 175cm^ tissue culture flasks (Gibco). For suspension culture experim ents, cells w ere cu ltu red in 10cm diam eter tissue culture petri dishes (Falcon) w hich had been sm eared w ith high vacuum silicon grease (Beckman). All cell w ashing and centrifugation steps w ere carried out in 27ml universal tubes (Sterilin).

2.2.2 Cell lines and Secondary Cell C ultures

O rigin/Species Cell Type S upplier

Swiss 3T3 M ouse Fibroblast ICRF

3T3 SV40 M ouse V irally transform ed fibroblast

ICRF

REF E15 rat embryo Secondary fibroblast

'in -h o u se '

T able 1: Cells used in these investigations.

2.2.3 C ell Passaging: The m edium from adherent cells w as rem oved from flasks by aspiration, cells w ere rinsed briefly w ith Ix Trypsin-EDTA

solution (Sigma) w hich w as then discarded and replaced w ith 5-10ml fresh trypsin-EDTA solution. The flasks w ere incubated at 37°C for 5-10 m inutes until all cells had dissociated from the substrate, and transferred to universal tubes containing an equal volum e of DMEM +10% ECS, to inactivate the

trypsin. The resultant cell suspension w as centrifuged in an MSE benchtop at 150g for 10 m inutes at room tem perature. Pellets w ere resuspended gently in fresh m edium and counted using a N eubauer haem ocytom eter. Cells w ere replated at the required concentration, depending on the experim ent and cell type involved. All cultures w ere grow n in a 37°C h u m id ified in cu b ato r (LEEC) in an atm osphere of 5% CO2 and 100% hum idity.

2.2.4 Cell Storage; Freezing and T haw ing: After harvesting cells w ere counted, pelleted at 150g for 10 m inutes and resuspended in 90% ECS w ith 10% DMSO (Sigma, cell culture grade) at a concentration of 1x10^ cells/m l. 1ml aliquots w ere transferred to 1.5ml screw top freezing vials (Nunc) w hich w ere insulated and cooled slowly to -85°C over a 24 h our period. Cells w ere stored u n d er liquid nitrogen (-196°C) indefinitely. Cells w ere thaw ed rapidly in a 37°C w aterbath, transferred to universal tubes containing DMEM + 10% ECS, pelleted at 150g for 10 m inutes, then resuspended and replated as above.

2.2.5 R at Em bryo F ibroblast (REF) Production: Secondary REEs w ere used for in this study. These were derived from sterile dissection of 15 day old rat em bryo limb-buds. The limb buds were rem oved using sterile scissors and forceps and rinsed in an excess of DMEM + 10% ECS. The limbs w ere teased a p a rt by sh earin g w ith tw o 2 1 gauge needles u n til no lu m p s of tissue

rem ained. The resulting cell suspension w as tran sferred to 175cm^ flasks containing DMEM +10% ECS and incubated at 37°C until confluent. The cells w ere then passaged, and m aintained in flasks or frozen for storage. Cells w ere generally discarded betw een passage num bers 10 and 15.

2.3 A ntibodies

2.3.1 Anti-C4 M onoclonal A ntibody: The anti-C4 m onoclonal antibody w as raised against chicken gizzard hom ogenate (Lawson, 1983). Briefly, the g izzard hom ogenate w as m ixed w ith F re u n d 's ad ju v an t an d rep eated ly injected into B alb /c mice over an eighty-seven day period at w hich p o in t extracted lym phocytes w ere fused w ith im m ortal SP2 cells, selected and then c lo n e d b y lim itin g d ilu tio n . T hose clo n es p ro d u c in g s ig n ific a n t im m u n o flu o rescen t p attern s in perm ealised 3T3 cells w ere selected an d frozen for further characterisation (Lawson, 1983).

2.3.2 Anti-C4 Polyclonal A ntibody: The anti-C4 polyclonal antibody w as generated by Shapland et al. (1993) from a partially purified fraction of sheep a o rta w h ich w as d ialy sed o v e rn ig h t into 50mM Tris b ase, p H 7.5, electrophoresed on 3mm thick 12% SDS-PAGE gels and the band containing transgelin excised and rinsed in Tris base, p H 6.8. The ban d (cut into sm all

pieces) w as then electroeluted into Ix Laemm li ru n n in g buffer (25mM Tris base, 192mM glycine, 0.1% (w /v ) SDS) in dialysis tubing (Spectra/por), acetone precip itated and injected into rabbits over a three m on th period. A DEAE (W hatm an) p rep a red IgG fraction w as p assed over an affinity colum n of p u rified tran sg elin (2mg) coupled to Affigel (BioRad), elu ted w ith 50mM d iethylam ine (Sigma), p H 11.5, im m ediately neu tralised w ith IM Tris.HCl (pH 7.5), dialysed into PBS and concentrated by m illipore filtration.

2.4 Im m unofluorescence

This m ethod is included by kind perm ission of D r D. Lawson, and w as carried out by him.

2.4.1 Labelling of Cytoskeletons: REF cells were plated onto uv sterilised 13mm diam eter sterile coverslips (BDH) in 24-well dishes (Falcon) at a

perm eabilised in 0.1% TritonX-100 in 50mM KCl, 4mM M gCl2, Im M EGTA,

lOmM im idazole pH7, Im M NaNg (Sigma). Cells w ere rinsed, p lu n g ed into m ethanol at -20°C, rehydrated, incubated in block buffer (0.3% BSA, lOOmM lysine in PBS) and probed w ith m ouse anti-C4 m onoclonal an tibody at a concentration of SOpg/ml in PBS + block buffer. Follow ing this they w ere in cu b a te d w ith goat an ti-m o u se IgG specific a n tib o d y co n ju g ated to rhodam ine (Cappel Laboratories) at a concentration of 5 0 |ig /m l and incubated in a 1 : 1 0 dilution of phalloidin/fluorescein, rinsed and m ounted onto glass

slides in 20% gelvatol (Polyvinyl alcohol, supplied by M ontsanta Polym ers). C overslips w ere visualised on a N ikon O p tip h o t fluorescence m icroscope w ith 60x objective and lOx eyepiece.

2.4.2 D e te rg e n t E xtraction: REEs w ere p lated on coverslips and perm eabilised as above. Cells were extracted in Buffer P + /- 60mM KCl + 0.5% CHAPS or 0.1% Saponin, rinsed, p lu n g ed into m eth an o l at -20°C, th en reh y d rated The cells w ere then incubated in block buffer (as above), probed w ith 2 0 |ig /m l m onoclonal anti-C4 antibody, rinsed and incubated w ith IgG- specific goat anti-m ouse rhodam ine at a dilution of 1 in 1 0 0 in block buffer.

Cells w ere then incubated in a 1:10 dilution of phalloidin/fluorescein, rinsed, m ounted onto glass slides in 2 0% gelvatol and visualised as above.

2.5 Sam ple Preparation for SDS-PAGE

2.5.1 T otal Cells: All cells used for these experim ents w ere cultured as described in Sections 2.2.1/2.2.3 until alm ost confluent, before harvesting.

Cells w ere transferred into a universal tube, w ashed in 25ml Ix PBS, pelleted by centrifugation in an MSE benchtop at 150g for 10 m inutes, resuspended in 1.5 m l PBS w ith protease inhibitors (see section 2.5.3) and transferred to a 1.5ml m icrofuge tube. After pelleting by centrifugation (6000g for 20 seconds

in an MSE M icroCentaur) the final pellet w as resuspended in two volum es of Laemmli sam ple buffer and boiled for 3 m inutes before freezing at -20°C.

2.5.2 N u clear P rep aratio n b y D eterg en t Extraction: Intact cells w ere processed as in Sections 2.2.1 and 2.2.3 an d a control sam ple rem oved,

resuspended in laemmli sam ple buffer and frozen. N uclei w ere isolated from rem aining intact cells by extraction w ith one the follow ing detergents w hich perm eabilise cellular m em branes.

(a) 0.5% CHAPS (Boehringer M annheim). (b) 0.1% Saponin (Sigma).

(c) 0.1% N onidet P-40 (Sigma). (d) 1% Triton X-100 (Sigma).

C ells w ere gently resu sp en d ed in 2 volum es of the req u ire d d e te rg en t

solution in PBS w ith protease inhibitors (see section 2.5.3) and incubated on ice for 20 m inutes. To separate the insoluble nuclear pellet from the soluble cytoplasm ic com ponent the sam ple w as th en p elleted b y cen trifu g atio n (13,000g, MSE M icroCentaur, for 15 m inutes at 4“C). The soluble su p ern atan t w as then rem oved using a draw n pasteur pipette and transferred to a fresh 1.5ml m icrofuge tube. The nuclear pellet w as resuspended in 2 volum es of Laemmli sam ple buffer and lOOpl of Laemmli sam ple buffer w as ad d ed to the su p ern atan t. Both sam ples w ere boiled for 3 m inutes before freezing. The sam ples w ere then ru n on a test m ini gel w hich w as subsequently stained in Coomassie blue and destained (see section 2.9.1).

2.5.3 Protease In h ib ito rs: All cell extractions w ere carried out in PBS containing a spectrum of protease inhibitors w hich w ere m ade as stock

solutions in d d H2 0, aliquoted and stored at -20°C (unless otherw ise stated).

In h ib ito r Target W orking Cone. Stock

AEBSF (Melford) serine proteases Im M lOOmM A p ro tin in (Sigma) serine proteases 2p g /m l 1 2m g /m l B enzam idine (Sigma)

throm bin and trypsin 1 5 |ig /m l 1 5 m g /m l C hym ostatin (Sigma) chym otrypsin 2p g /m l 5 m g /m l in DMSO EDTA (Fluka) m etalloproteases 0.2mM 0.5M, RT L eupeptin (Sigma)

serine & thiol 2|ig /m l 5 m g /m l

P epstatin A (Sigma) serine proteases 2p g /m l 5 m g /m l in m e th a n o l

T able 2: Protease Inhibitors.

2 . 6 Cell Cycle A rresting

Cells w ere cultured as in Sections 2.2 . 1 and 2.2.3, and treated w ith m etabolic

inhibitors or changes in culture conditions to arrest them at distinct phases in the cell cycle.

2.6.1 Serum Starve (blocks in Go/Gi): C ultured cells (in DMEM + 10% ECS) w ere aspirated, w ashed in serum free DMEM and refed w ith DMEM + 0.2% ECS for 48 hours (Lamb et al., 1990).

2.6 . 2 L ovastatin (blocks in Gi): Lovastatin (Merck, Sharp and D ohm e

R esearch Pharm aceuticals) w as first converted from its inactive lactone p ro d ru g form to its active dihydroxy open acid from by dissolving 52mg in 1.04 ml ethanol (95%) and adding SlSpl of IN NaOH. The resulting solution w as neutralised w ith IN HCl to pH 7.2 and brought to a volum e of 13ml w ith d d H2 0 . This stock (lOmM) w as then aliquoted and frozen. L ovastatin w as

a d d ed to m edium to a concentration of 20|xM and cells incubated w ith the d ru g for 36 hours (Keyomarsi et al., 1991).

2.6.3 A p h id ico lin (blocks in G i/S): Cells w ere cu ltu red in m edium containing aphidicolin (Sigma) at a concentration of I p g /m l, for 24 hours (Golsteyn et al., 1995).

2.6.4 D o u b le T h y m id in e Block (blocks in S): Cells w ere cultured to alm ost confluence then cultured w ith m edium containing 2mM thym idine (Sigma) for 24 hours, asp irated , w ashed in th y m id in e free m ed iu m and c u ltu re d in th y m id in e free m ed iu m for a fu rth e r 24 h o u rs. M ed iu m containing 2mM thym idine w as again ad d ed and the cells incubated for a final 24 hours (Golsteyn et al., 1995).

2.6.5 N ocodazole (blocks in M): Cells w ere cultured for 48 h o u rs in m edium containing nocodazole (Sigma) at a concentration of O .lp g /m l (Poon et al., 1995).

2.6 . 6 Post arresting: Following cell cycle arresting treatm ents, the cells

w ere h arv ested b y trypsinisation (see section 2.2.3) an d counted u sin g a N eu b au er H aem ocytom eter. Ix 10^ cells w ere pelleted by centrifugation, the m edium drained off and vortexed in 1ml -20°C 70% ethanol for fixing. Fixed

sam ple w ere stored at -20°C for analysis by FACS. The rem aining cells w ere separated into a control total cell sam ple and a sam ple for detergent extraction (see sections 2.5.1/2.5.2).

2.6.7 P ro p id iu m Iodide Staining and FACS analysis: Fixed Cells w ere p e lle t by cen trifu g a tio n , the su p e rn a ta n t rem o v ed an d sta in e d w ith p ro p id iu m iodide (PI, a DNA specific dye) containing stain solution (0.2 m g /m l PI (Sigma), 5 m g /m l RNase (DNase free. Sigma) in PBS and incubated for 30 m inutes in the dark. Cells w ere analysed by a Coulter EPICS Elite Flow cytom eter (Coulter Electronics) using C oulter w orkstation softw are, version 4.0. Cells w ere illum inated w ith a 488nm argon ion laser and fluorescence d ata was collected betw een 565 and 585nm. Traces displayed show a profile of at least 1 0 , 0 0 0 cells and low level discrim ination was used to discard data from

cell debris. By staining DNA w ith propidium iodide the DNA content of cells can be m ea su re d and as DNA co n ten t v aries d u rin g the cell cycle, m easurem ent of relative DNA content by FACS analysis can q uantitate the percentage of cells w hich are blocked in each phase. The FACS m achine is unable to sort cells in Gi from those in Go, or those in G2 from those in M.

2.7 N uclear Solubilisation

To so lu b ilise n u clear u ltra stru c tu re the fo llo w in g co n d itio n s for cell extraction w ere used, alw ays in the presence of protease inhibitors (Section 2.5.3).

2.7.1 Nuclease D igestion

(i) D N ase I (0.25m g/m l) (Sigma) and RN ase A (0.25m g/m l) (Sigma) in PBS w ith 0.5% TritonX-100 (Mirzayan et al., 1992)

(ii) DN ase I (0.25mg/ml) and RNase A (0.25m g/m l) in D igestion B uffer lOmM PIPES, p H 6.8, 50mM N aCl, 300mM sucrose, 3mM EGTA, 0.5%

TritonX-100 (all Sigma), w ith DNasel (0.25m g/m l) and RNaseA (0.25 m g /m l), (He et al, 1990; M irzayan et al., 1992).

2.7.2 Salt Extraction

(i) 0.2M NaCl (Sigma)+ D igestion Buffer (Mirzayan et al., 1992)

(ii) l.OM NaCl + D igestion Buffer (Mirzayan et al., 1992)

(iii) 2.0M NaCl + D igestion Buffer (Mirzayan et al., 1992)