G1/S staged(hydroxyurea)

G2/M staged(nocodazole)

A B

2n 4n

2n 4n

G1/S

ORC1

Cdt1

MCM2

Actin

HBO1

G2/M MW(bp)

100

100

200

100

300ORC2

ORC1

Cdt1

HBO1

Whole cellular extracts

Chromatinfractions

Asy. G2/MG1/S Asy. G2/MG1/S

Cdc6

Supplementary Figure 3

Levels of mRNA and protein bound to bulk chromatin at different stages of the cell cycle, in response to actinomycin D treatment, and upon HBO1 overexpression. (A) Expression level of HBO1, MCM2, ORC1, and Cdt1 mRNAs in CCL-156 cells staged in early S phase (hydroxyurea block) or at the G2/M transition (nocodozole block). Efficient staging was confirmed by FACS analysis. (B) Western blot analysis of HBO1, Cdt1, Cdc6, ORC1, and ORC2 expression in CCL-156 cells staged in early S phase and at the G2/M transition. (C) Expression level and chromatin association of HBO1 and replication factors in the absence or presence of actinomycin D (actD). Note that, for unknown reasons, soluble HBO1 is degraded following ActD treatment, whereas the level of chromatin-bound HBO1 is unaffected. (D) Expression level and chromatin association of HBO1 and replication factors under conditions of HBO1 overexpression. Values for each protein indicate fold-difference upon HBO1 overexpression

C

ORC1

ORC2

MCM3

HBO1

Cdt1

Actin

Whole cellular extracts

- + - +

Cdc6

Chromatinfractions

Actinomycin D

D

MCM3

MCM5

ORC1

Cdt1

Cdc6

Myc-HBO1

HBO1

Myc-HBO1control

Whole cellular extracts

Chromatinfractions

- ++ -

- ++ -

1.2

1.1

0.98

0.97

1.1

-

3.5