HBO1 siRNA #2

1. Supplementary Figure 1 A E C HeLa cells control siRNA Whole cell extracts Chromatin fractions Input (5%) IP : HBO1 IP : IgG %G1/G0 %S %G2/M 41.70 26.56 31.74 - - - - + + HBO1 siRNA #2 200 Number of cells - - - - + + Control siRNA 100 ORC1 HBO1 2n 4n ORC1 MCM2 Cdc6 HBO1 siRNA #2 Cdt1 HoxA10 %G1/G0 %S %G2/M 24.76 41.78 33.46 Geminin 200 Gcn5 Sp1 Number of cells 100 MCM2 EZH2 4n 2n MCM3 MCM Complex MCM4 B D Su(z)12 MCM5 MCM6 % BrdU positive cells HBO1 siRNA #1 HBO1 siRNA #2 siRNA control Input (50%) MCM7 40 30 * 20 10 HBO1 control control siRNA HBO1 siRNA #2 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.