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Supplementary Materials and methods
Cell culture and cell cycle analysis
The bladder carcinoma cell line, 5637, which lacks RB gene and p53 function (RIKEN Bioresource Center Cell
Bank) was cultured in RPMI 1640 containing 10% fetal calf serum (FCS). Human normal lung fibroblasts
(WI-38), human foreskin fibroblasts (HFFs), Rat embryonic fibroblast cell line (REF52), a cervical carcinoma cell
line (C33-A) with mutant pRb and p53 and the osteosarcoma cell line (Saos-2), which lacks the RB gene and p53
function (American Type Culture Collection), were cultured in Dulbecco’s modified Eagle medium (DMEM)
containing 10% FCS. WI-38 cells were rendered quiescent by serum-starvation for 72 h and then restimulated with
a 10% final concentration of FCS to allow re-entry into the cell cycle. For cell cycle profiling, cells at specific time
points were stained with propidium iodide and analyzed by FACS as described (Ohtani et al., 2000). Wild type and
an EREA mutated p14ARF promoter constructs were integrated into the genomic DNA of REF52 cells using
ViraPower lentiviral Expression System (Invitrogen). Integrated cell populations were selected with blasticidin for
10 days.
Plasmid construction
pARF-Luc(-736), pCDC6-Luc/wt, pCDC6-Luc/mt, pE2/wtx4-Luc and pE2/mtx4-Luc were generated by
subcloning promoters into pGL3-Basic (Promega) from pExon1-Luc
(a gift from G. Peters),
pHsCDC6-Luc(-570), pHsCDC6-Luc(-E2F), pE2WTx4-Luc and pE2MTx4-Luc, respectively (Bates et al., 1998;
Ohtani et al., 1998; Ohtani et al., 2000). We generated 5’ deletion constructs of pARF-Luc(-736) and an internal
deletion construct of the region –231~-150, pARF-Luc/dl(-231~-150) by digestion with the restriction enzymes.
Other deletion constructs of the region (-231~-150) and point mutation constructs of pARF-Luc/dl(-204~-187),
were generated by inserting double-stranded oligonucleotides (Figure 2B and 2D). The plasmid
pARF-Luc(-736/mt) was generated by site-directed mutagenesis, which changed the same nucleotides as those of
mutant 5. Three copies of the EREA fragment were joined upstream of the SV40 core promoter in pGL3-Promoter
(Promega) or the p14ARF core promoter in pARF-Luc(-66) to generate pEREAx3-Luc and pEREAx3-(-66)-Luc,
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respectively. Four copies of the E2 enhancer were fused upstream of the p14 ARF core promoter in pARF-Luc(-66),
generating pE2/wtx4-(-66)-Luc. The E2F1 expression plasmid, pDCE2F, the Cyclin D1 expression vector, pR1,
the cdk4 expression vector, pCMVcdk4, the -galactosidase expression vector, pCMV--gal, the estrogen receptor
fused E2F1 and E2F1 mutant vectors, pBabe HAERE2F1 and pBabe HAERE132 (gifts from K. Helin), the
p16INK4a expression vector, pCMVp16, the pRb expression vector, pSVhRB, the mutant pRb expression vector,
pSVhRB J82M, the constitutive active mutant pRb expression vector, pPSM.7-LP (a gift from E. Knudsen), the
adenovirus 12S E1a expression vector and E1a mutant vectors have been described (gifts from M. Ikeda)
(Alevizopoulos et al., 1998; DeGregori et al., 1995; Heibert et al., 1992; Johnson et al., 1994; Knudsen and Wang,
1997; Muller et al., 2001).
Transfection and reporter assay
Luciferase assays proceeded as described (Ohtani et al., 1998). Expression and reporter plasmids were transfected
into cells by lipofection together with the internal pCMV--gal control that monitored transfection efficiency.
Luciferase activities were assayed using the Luciferase Assay System (Promega) and normalized to
-galactosidase activities. All assays were performed three times in duplicate and values are shown as means±SD.
Chromatin immunoprecipitation (ChIP) assay
The ChIP assay proceeded as described (Takahashi et al., 2000). PCR was performed with -32P-dCTP and
individual specific primer sets: the p14ARF promoter, 5’-GGTTTGCTCTTT GATGCTCTTGAT-3’ and
5’-ACTTTCGGGTGGGGTGTAA-3’, the human -actin promoter, 5’-GGTTTGCTCTTTGATGCTCTTGAT-3’
and 5’-ACTTTCGGGTGGGGTGTAA-3’, ARF-Luc(-736), 5’-GGTTTGCTCTTTGATGCTCTTGAT-3’ and
5’-CTTTATGTTTTTGGCGTCTTCC-3’, the rat -actin promoter, 5’-GAGCGAGCCGAGCCAATCA-3’ and
5’-CCACACCGCGGGAATACGACTG-3’, the rat DHFR promoter, 5’-CTTAGGCTTCCCGCAGACTTGA-3’
and 5’-CATATTTTGGGACACGGCGACGAT-3’, and the specific primer set for the CDC6 promoter as described
(Takahashi et al., 2000). Anti-E2F1 (sc-251 X), anti-E2F3 (sc-878X), anti-E2F4 (sc-512 X), anti-p130 (sd-317 X),
and anti-HA (sc-804) (All from Santa Cruz) antibodies were used for immunoprecipitating protein-DNA
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complexes. Anti-HA antibody was the control. The PCR products were separated by electrophoresis in 8%
acrylamide gels. Radioactivities of the signals were measured with an image analyzer BAS 1500 (Fuji Film).
RNA interference
A 21-bp DNA sequence for RNA interference against the human RB gene (nucleotides 548-568 of the human RB
cDNA; GenBank accession number M33647) was selected based on a published report (Elbashir et al., 2002). The
shRNA expression vector, pshRB was generated by inserting double-stranded oligonucleotides including the target
sequence into the expression vector, pSilencer 2.0-U6 (Ambion), according to the manufacturer’s protocol.
Negative control vector or shRNA expression vector was introduced into 293 and WI-38 cells by lipofection. The
effect of pshRB was examined by immunoblotting and reporter assays respectively.
Immunoblotting
Aliquots of whole cell lysates separated by SDS-PAGE, were blotted onto an Immobilon-P membrane (Atto), and
then incubated with antibodies specific for individual protein. Antibodies for immunobloting were p14 ARF (Ab-1;
NeoMarkers), E2F1 (sc-251 X; Santa Cruz), pRb (sc-50; Santa Cruz), E1a (M58; BD Biosciences Pharmingen), or
-tubulin (CP06; Oncogene Research Products). Blots were incubated with HRP-conjugated secondary
antibodies, and proteins were detected by ECL plus Western Blotting Detection System (Amersham Biosciences)
according to the manufacturer’s protocol.
Infection with recombinant adenoviruses
WI-38 cells and REF52 cells were infected with recombinant Ad-E2F1 expressing E2F1 or control Ad-Con at
multiples of 150 or 100 plaque forming units per cell as described (Schwarz et al., 1995). Ad-12SE1A was
generated from pCMV-12SE1A using ViraPower Adenoviral Expression System (Invitrogen) according to the
supplier’s protocol.
Isolation of mRNA and RT -PCR
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Total RNA was extracted using Isogen (Nippon Gene) and poly (A)+ RNA was purified using PolyA Tract
(Promega) according to the manufacturer’s protocol. First strand cDNA was synthesized using a 1st Strand cDNA
Synthesis Kit for RT-PCR [AMV] (Roche) according to the supplier’s protocol. The following individual specific
primer
sets
were
used
for
5’-ACTTTCGGGTGGGGTGTAA-3’,
PCR:
CDC6,
GAPDH,
5’-GGTTTGCTCTTTGATGCTCTTGAT-3’
5’-GGAGTCCACTGGCGTCTTCA-3’
and
and
5’-GAGGGGCCATCCACAGT CTT-3’, and those for p14ARF were as described (Ohtani et al., 2000). The PCR
products were resolved by electrophoresis on 2% agarose gels and visualized by ethidium bromide staining.
Northern hybridization and Southern hybridization
mRNA was prepared from REF52 cells expressing ER-E2F1 or ER-E132. Gel electrophoresis, transfer to nylon
membranes and hybridization proceeded as described (Ohtani et al., 1998). Genomic DNA were prepared from
REF52 cell lines and digested by HindIII and XbaI. A luciferase probe was excised from the pGL3-Basic vector
and the internal control -galactosidase probe was excised from pgal-Basic (Clontech). Radioactive signals were
measured using an image analyzer BAS 1500.
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Supplementary References
Alevizopoulos, K., Catarin, B., Vlach, J. and Amati, B. (1998) A novel function of adenovirus E1A is required to
overcome growth arrest by the CDK2 inhibitor p27 Kip1. EMBO J, 17, 5987-5997.
DeGregori, J., Leone, G., Ohtani, K., Miron, A. and Nevins, J.R. (1995) E2F-1 accumulation bypasses a G1 arrest
resulting from the inhibition of G1 cyclin-dependent kinase activity. Genes Dev, 9, 2873-2887.
Elbashir, S.M., Harborth, J., Weber, K. and Tuschl, T. (2002) Analysis of gene function in somatic mammalian
cells using small interfering RNAs. Methods, 26, 199-213.
Johnson, D.G., Ohtani, K. and Nevins, J.R. (1994) Autoregulatory control of E2F1 expression in response to
positive and negative regulators of cell cycle progression. Genes Dev, 8, 1514-1525.
Knudsen, E.S. and Wang, J.Y. (1997) Dual mechanisms for the inhibition of E2F binding to RB by
cyclin-dependent kinase-mediated RB phosphorylation. Mol Cell Biol, 17, 5771-5783.
Ohtani, K., Iwanaga, R., Arai, M., Huang, Y., Matsumura, Y. and Nakamura, M. (2000) Cell type-specific E2F
activation and cell cycle progression induced by the oncogene product Tax of human T-cell leukemia
virus type I. J Biol Chem, 275, 11154-11163.
Schwarz, J.K., Bassing, C.H., Kovesdi, I., Datto, M.B., Blazing, M., George, S., Wang, X.F. and Nevins, J.R.
(1995) Expression of the E2F1 transcription factor overcomes type  transforming growth
factor-mediated growth suppression. Proc Natl Acad Sci USA, 92, 483-487.
Takahashi, Y., Rayman, J.B. and Dynlacht, B.D. (2000) Analysis of promoter binding by the E2F and pRB families
in vivo: distinct E2F proteins mediate activation and repression. Genes Dev, 14, 804-816.
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Supplementary Figure legends
Figure S1. Induction of p14ARF protein by the ectopic expression of E2F1. Cell extracts were from asynchronously
growing WI-38 cells infected with either Ad-E2F1 or Ad-Con. Protein expression was detected by
immunoblotting. Internal control was -tubulin.
Figure S2. Activation of the EREA reporter by ER-E2F1 in REF52 cells. REF52 cells were transfected with the
EREA reporter {EREAx3-(-66)-Luc} or control reporter {ARF-Luc(-66)} plasmid (1 g), expression vector for
ER-E2F1 (100 ng) and pCMV--gal (1 g) as internal control. Transfected cells were cultured for 2 days,
incubated with hydroxytamoxifen (OHT) and/or cycloheximide (CHX) as indicated, and harvested at 8 h later.
ER-E132 is a mutant E2F1, which lacks binding ability to DNA.
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