G1/S staged

1
Supplementary Figure 3
A
B
G1/S
G2/M
MW(bp)
Chromatin fractions
Whole cellular extracts
HBO1
100
2n
4n
Asy.
G2/M
G1/S
Asy.
G2/M
G1/S
G1/S staged (hydroxyurea)
ORC1
HBO1
100
Cdt1
Cdt1
200
ORC1
MCM2
100
Cdc6
2n
4n
G2/M staged (nocodazole)
300
Actin?
ORC2
C
Whole cellular extracts
Chromatin fractions
Actinomycin D
-

-

Cdt1
HBO1
ORC1
ORC2
Cdc6
MCM3
Actin?
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