Page 1 \ ?^p 6r.1 CELL CYCLE CONTROL OF HUMAN H4 ...

Aziz F: Cen ddo Contot ol H4 Gere l@sqiol@l
adhesion to the plales andto Prevern the Peeling ol lhe cells from lhe
plai€. Similany, nOB cells wero plated at a densily ol0 4xlCF celb per
1oo mm plate in minimal ess€nlial m€dium (MEM) containing Ea e s
salt and supplemented wiih 10% l6tal call serum 100 units ol
penicillin/mJ, 1Oo ,rglml stfeptomvcin and 2 mM L-glllamine Th€ cen3
were microscopcally inspeded belore transfection ro ensure thal cols
are actLvely prolilerating (ie.,40 _ 50% confluent) and devoid or
microbial conlamination For esch 1oo millimeter plste lranslection
mdures were prepared in 2 o ml cenlrillge tubes serum'tre media
(1.5 ml)was combinedwith 1.5ploi chloroquine (slgmaistock soluton
oisO mg/mland slored al -20'C n a daA containe4 afd 6 ! oroEAE
Dsxlran (Pharmaciarstock soution ol50 rng/mland stored al4'C) to
nnal conc€nlrations ol oo5 mg /ml chloroquln i'd 0 2 mg/mr DFAE
Dextran. The mlxtlres w€r€ complemenled wilh 20 rg of plasm d DNA
in a minimal voLume ol TE bufler (ie up ro 10 ,rl)
Plales were wasneo
twice wilh PBS, and theeLlswere covered with the 1 5 mloNA mi*ure
lolLowed by i.cubar on fof 2 5 lo 3 holrs al 37'C After lhis pe' od' the
DNA was aspraled from lhe pates and the cels were subjected io
glycerolshock byth€ addilion oi 10% glyc€rolin serumJree med a and
incubatio. for exaclly 90 sec.nds The glycerol 'nixu'e was removed
by aspkation, the plates rete vvashed tlvice with Pgs and elB were
reled with complele nedia- The plales were incubat€d ior 4a lo 72
hours at 37'C, and cells were hatuested as describ€d above tor Hela
70