G1/S staged (hydroxyurea)

1. D Whole cellular extracts Chromatin fractions - + + - Myc-HBO1 - + + - control 1.2 1.1 0.98 0.97 1.1 - 3.5 MCM3 MCM5 ORC1 Cdt1 Cdc6 Myc-HBO1 HBO1 Supplementary Figure 3 A B G1/S G2/M MW(bp) Chromatin fractions Whole cellular extracts HBO1 100 2n 4n Asy. G1/S G2/M Asy. G1/S G2/M G1/S staged (hydroxyurea) ORC1 HBO1 100 Cdt1 Cdt1 200 ORC1 MCM2 100 Cdc6 2n 4n G2/M staged (nocodazole) 300 ORC2 Actin C Chromatin fractions Whole cellular extracts 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