HBO1 regulates BrdU incorporation, cell cycle progression and MCM complex association with chromatin. (A) Detection of HBO1 association with ORC1 and MCM2 by co-immunoprecipitation in HEK293 cells. (B) Impact of siRNA contructs on HBO1 protein expression level. (C) Cell cycle distribution of HEK 293 cells 72 hours after treatment with a control siRNA (top) or HBO1 siRNA#2 (bottom). (D) Level of BrdU incorporation in cells treated with HBO1 siRNA#2 and control siRNA 30 minutes after addition of BrdU in the culture media (n=4). *, p<0.05. (E) Western blot analysis of ORC1, Cdc6, Cdt1, Geminin, GCN5 and MCM2-7 proteins expression and chromatin association in HBO1 depleted cells. The figure is representative of 3 independent experiments.

D

HBO1 siRNA #2 +-

HBO1

ORC1

MCM3

MCM4

HeLa cellsWhole cell

extractsChromatin fractions

MCM5

MCM6

Gcn5

MCM2

MCM7

Cdc6

Cdt1

Geminin

Control siRNA-- + -

+--- + -

MCMComplexB

% BrdU positive cells

control controlsiRNA

HBO1 siRNA #2

10203040

*

200

%G1/G0%S %G2/M

2n

%G1/G0%S%G2/M

4n

4n2n

100

HBO1 siRNA #2

200

100

Num

ber

of c

ells

control siRNA

41.7026.5631.74

24.7641.7833.46

C

Num

ber

of c

ells

Supplementary Figure 1In

pu

t (5

0%)

HB

O1

siR

NA

#1

HB

O1

siR

NA

#2

siR

NA

co

ntr

ol

HBO1

EIP

: H

BO

1

IP :

IgG

Inp

ut

(5%

)A

Su(z)12

HoxA10

MCM2

ORC1

Sp1

EZH2