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mice ( e2f1 , e2f2 , e2f3 , ah - cre and rb ) used for the studies were in mixed background ( 129svev , c57bl/6ntac and fvb / ntac ) .
-napthoflavone ( sigma ; n3633 - 5 g ) was administered into 2 month old ah - cre mice three times within 24 hours as described previously14 and mice were harvested 7 or 90 days later .
-napthoflavone was also injected into pregnant female mice at 15.5 days postcoitum for analysis of embryos at e18.5 .
three independent samples from each genetic group were used for gene expression analysis by affymetrix microarray .
analysis of gene expression data were performed using brb - array tools developed by dr .
gene ontologies were predicted by david ( database for annotation , visualization and integrated discovery ) bioinformatics resources at the national institute of allergy and infectious diseases , nih .
x - gal staining , real - time rt - pcr , brdu , chip and tunel assays were performed as previously described8 , 19 .
primers for chip , real - time rt - pcr and genotyping are listed in supplementary fig .
supplementary figure 1 . deletion of e2f1 - 3 does not impair es cell growth or differentiation .
a. pcr genotyping of multiple es cell clones after electroporation with a cre - expressing plasmid driven by the elongation factor-1 promoter ( ef1-cre ) .
b. expression levels of nanog , oct4 , eomes and cdx2 were measured by quantitative rt - pcr in trophoblast stem ( ts ) cells and es cell clones .
consistent with the pluripotency of es cells derived from e2f1;e2f2;e2f3 embryos , these cells expressed high levels of oct4 and nanog but low levels of cdx2 and eomes ( ts cell markers ) .
es cells were derived from blastocysts at e3.5 from crosses between e2f1 ; e2f2 ; e2f3 and e2f1 ; e2f2 ; e2f3 mice .
colonies that grew from disaggregated blastocysts with es cell - like morphology were subcultured for expansion and were genotyped by pcr for e2f1 , e2f2 , and e2f3 .
e2f1 ; e2f2 ; e2f3 clones derived from two different embryos were expanded and electroporated with ef1-cre .
cells were plated at low density after electroporation and colonies were collected for genotyping and further culture .
approximately 55% to 70% of the resulting clones underwent recombination of the conditional e2f3 allele without any form of selection .
finally , es cells were checked for expression of es cell - specific and trophoblast stem ( ts ) cell - specific markers to verify their identities .
a. real - time rt - pcr analysis of e2f1 , e2f2 , e2f3a , and e2f3b expression in wild type mefs and es cells ( tc-1 line ) . b.
the tc-1 wild type es cell line expresses equivalent levels of e2f3 protein as detected by western blots probed with anti - e2f3 .
e2f1 - 3 triply - deleted ( tko ) es cells are used as a negative control . c.
chromatin immunoprecipitation was done with anti - e2f3 antibodies , using control (  ) or cre - treated ( + ) e2f1;e2f2;e2f3 mefs ( left panel ) as well as dko and tko es cells ( right panel ) .
supplementary figure 3 . gene expression and e2f4/p130 mediated repression in dko and tko es cells .
a. real - time rt - pcr analysis of p53 target gene expression in control (  ) or cre - treated ( + ) e2f1;e2f2;e2f3 mefs ( left panel ) as well as dko and tko es cells ( right panel ) .
the binding of e2f4 ( b ) and p130 ( c ) to e2f target promoters in control (  ) or cre - treated ( + ) e2f1;e2f2;e2f3 mefs ( left panel ) as well as dko and tko es cells ( right panel ) was detected by chromatin immunoprecipitation with anti - e2f4 and anti - p130 antibodies , respectively .
a. pluripotency of es cell lines was measured by scoring the number of teratomas containing defined cell lineages ( neural , muscle , epithelial , skin and inflammatory ) from a total number of teratomas that developed in mice injected with one wild type es cell line ( tc-1 ) , two dko es cell lines , and two tko es cell lines . b. growth rates of teratomas in ( a ) were calculated as the number of days required for teratomas to reach a volume of 200 mm .
ki67 ( a ) and tunel ( c ) staining of various organs from e9.5 e2f1;e2f2;e2f3 ( sko ) , e2f1;e2f2;e2f3 ( dko ) and e2f1;e2f2;e2f3 ( tko ) embryos .
quantification of ki-67-positive ( b ) and tunel - positive ( d ) cells from a minimum of three embryos of each genotype ( sko , dko and tko ) ; at least three sections for each embryo were analyzed .
western blots of protein lysates derived from wild type crypts and villi were probed with antibodies specific for e2f1 , e2f2 , and e2f3 .
villi lysates derived from ah - cre;e2f1;e2f2;e2f3 small intestines ( tko ) were used as a negative control ; note that a small amount of spill - over occurred from the adjacent lane .
a. 
 in situ x - gal staining was used to examine the expression of cre - recombinase in the small intestines of rosa26 ( control ) and ah - cre;rosa26 ( ah - cre ) mice after one , two and three days following -napthoflavone ( -nf ) injection .
deletion of e2f3 was measured by southern blot ( b ) and pcr ( c ) of dna isolated from small intestines with the indicated genotypes . d.
real - time rt - pcr was used to measure e2f4 - 8 expression in villi from control and tko small intestines .
a. h&e stained sections of small intestines from e2f1;e2f2;e2f3 ( control ) and ah - cre;e2f1;e2f2;e2f3 ( ah - cre ) pups that were harvested 3 and 24 days following -nf administration into pregnant females ( at 15.5 d.p.c . ) .
b. h&e stained sections of small intestines from control and ah - cre mice harvested 7 and 90 days following the administration of -nf into 2 month - old mice .
antibodies specific against cholecystokinin ( cck , red ) , somatostatin ( red ) , serotonin ( red ) and lysozyme ( red ) were used for identifying various differentiated endocrine cells in e2f1;e2f2;e2f3 ( control ) and ah - cre;e2f1;e2f2;e2f3 ( ah - cre ) small intestines .
supplementary figure 10 . increased -h2ax staining in e2f1 - 3 deficient small intestines a. immunohistochemical staining of -h2ax ( red ) and p - atm ( red ) in e2f1;e2f2;e2f3 ( control ) and ah - cre;e2f1;e2f2;e2f3 ( ah - cre ) intestinal crypts .
b. immunohistochemical staining of -h2ax ( red ) in wild type ( control ) and cre - treated e2f1;e2f2;e2f3 ( e2f1 - 3 tko ) retinas at various developmental stages indicated ( e14 , p0 and p18 ) . c. quantification of the -h2ax positive cells stained in ( b ) .
small intestine sections from e2f1;e2f2;e2f3 ( control ) and ah - cre;e2f1;e2f2;e2f3 ( ah - cre ( tko ) ) mice injected with -nf and harvested 7 days later were stained with antibodies for p53 .
b. tunel assays were performed on small intestine sections from e2f1;e2f2;e2f3;p53 ( control ) and ah - cre;e2f1;e2f2;e2f3;p53 ( ah - cre ( qko ) ) mice treated as in ( a ) .
heirarchical clustering analysis of differentially expressed genes ( > 1.5-fold and p<0.0001 ) in e2f1;e2f2;e2f3 ( control ) and ah - cre;e2f1;e2f2;e2f3 ( ah - cre ) villi and crypts is presented as a heat map ( a ) .
b. list of e2f target genes as defined by previous gene expression , reporter and chip assays17 , 18 .
expression of e2f - responsive genes is upregulated in e2f1 - 3 deficient small intestines and retinas .
a. quantitative real - time pcr was used to examine the relative expression of selected e2f - target genes in control and ah - cre crypts ( left panels ) and villi ( right panels ) using specific primers ( supplementary fig .
b. examination of mcm3 and pcna protein levels in e2f1;e2f2;e2f3 ( control ) and ah - cre;e2f1;e2f2;e2f3 ( ah - cre ) villi by western blot .
c. relative quantitative real - time pcr of a panel of genes in wild type and cre - treated p14 e2f1;e2f2;e2f3 ( tko ) retinas .
loss of rb in differentiating intestinal crypt cells ( crypt ) results in ectopic proliferation in villi .
a. cell extracts isolated from control crypts and villi were used to detect rb protein in western blots .
rbko , rb knockout crypt ; rb - p , hyperphosphorylated rb ; rb , hyporphosphorylated rb .
b. pcr based assay used to examine deletion of the rb allele from genomic dna purified from small intestines of rb , rb (  ) , ah - cre;rb ( + ) mice after 7 days of -napthoflavone ( -nf ) administration . c. small intestines from -nf - injected 2 month - old rb ( control ) and ah - cre;rb ( rbko ) mice
d. brdu analysis was performed in control and rbko intestines harvested at embryonic day 18.5 ( e18.5 ) and at 2 months of age ( p60 ) . for the embryonic analysis of the small intestine ( e18.5 )
e. quantification of brdu incorporation in villi and crypts of animals with the indicated genotypes and ages .
a. small intestine sections from rb ( control ) and ah - cre;rb ( rbko ) mice , which were injected with -nf and harvested 7 days after , were used for tunel staining .
alcian blue , goblet cells were identified by alcian blue staining ; other antibody - based markers for cholecystokinin ( cck , red ) , serotonin ( red ) , somatostatin ( red ) , lysozyme ( red ) and fatty acid binding protein ( fabp , green ) were used to identify endocrine cells , paneth cells , and absorptive cells , respectively .
a. sections of small intestines from rb ( control ) , ah - cre;rb ( rbko ) and ah - cre;rb;e2f1;e2f2;e2f3 ( qko ) mice that were harvested 7 days post--nf - injection were stained with h&e .
b. brdu analysis was performed in control , rbko and qko small intestines from mice treated as in ( a ) .
a. heatmap representation of heirarchical clustering analysis of differentially expressed genes in crypts and villi of rb ( control ) and ah - cre;rb ( rbko ) mice ( p<0.0001 ) .
three independent samples from each genetic group and tissue ( crypt / villus ) were analyzed .
b. scatter plots comparing the expression of known e2f - target genes in control and rbko crypt and villus . c. expression of e2f - target genes in control and rbko villi was examined by real - time rt - pcr .
error bars indicate standard deviation from triplicate samples . d. immunohistochemical staining of mcm3 ( green ) and pcna ( red ) in control , rbko and qko villi .
yellow dotted line outlines the luminal side of the villus ; white dotted line outlines the outer side of the villus .
hierarchical clustering analysis of differentially expressed genes in control , rbko ( rko ) , tko , qko villi ( p<0.0001 ) .
note that wild type ( con ) and rko samples segregate from tko and qko villi samples ; a similar separation of genetic groups was achieved when the associated crypts were analyzed in the same manner .
list of primers used for genotyping ( a ) and chip assays ( b ) . c. list of antibodies used in this study .
list of primers used for real - time rt - pcr analysis of gene expression .
in the classic paradigm of mammalian cell cycle control , rb functions to restrict cells from entering s phase by sequestering e2f activators ( e2f1 , e2f2 and e2f3 ) , which are invariably portrayed as the ultimate effectors of a transcriptional program that commit cells to enter and progress through s phase1 , 2 . using a panel of tissue - specific cre - transgenic mice and conditional e2f alleles 
 we examine the effects of e2f1 , e2f2 and e2f3 triple deficiency in murine es cells , embryos and small intestines . 
 we show that in normal dividing progenitor cells e2f1 - 3 function as transcriptional activators , but contrary to current dogma , are dispensable for cell division and instead are necessary for cell survival . in differentiating cells 
 they function in complex with rb as repressors to silence e2f targets and facilitate exit from the cell cycle . 
 the inactivation of rb in differentiating cells resulted in a switch of e2f1 - 3 from repressors to activators , leading to the superactivation of e2f responsive targets and ectopic cell divisions , and loss of e2f1 - 3 completely suppressed these phenotypes . 
 this work contextualizes the activator versus repressor functions of e2f1 - 3 in vivo , revealing distinct roles in dividing versus differentiating cells and in normal versus cancer - like cell cycles in vivo .