Human Orc2 localizes to centrosomes, centromeres and heterochromatin during chromosome inheritance - PubMed (original) (raw)

Orc2 depletion affects S-phase progression. (A) Immunolabeling of cells at 72 h after transfection with control or Orc2 siRNA duplexes (a, b); BrdU incorporation during a 24 h pulse (a′, b′); Nomarski images (a″, b″). Scale bar, 10 μ

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. (B) Luciferase (control) and Orc2 siRNA duplex-treated cells were subjected to biochemical fractionation (Méndez and Stillman, 2000). The relative abundance of different replication proteins was tested by immunoblotting. S2, soluble cytosolic fraction; S3, soluble nuclear fraction; P3, chromatin-enriched fraction. Antibodies against Orc2, Orc1, Orc3, Orc6, Cdc6, MCM2, MCM3, MCM4, MCM7 and PCNA were used. Cytosolic kinase MEK2 was used as a control. Note that Orc3 and PCNA loading was affected in Orc2-depleted cells. (C) Chromatin association of PCNA was assessed by IF in control (a–a′) and Orc2-depleted cells (b–b′). DNA was stained with DAPI (a′, b′). Scale bar, 10 μM. (D) Chromatin-bound MCM3 was analyzed by a detergent pre-extraction procedure in control (a) and Orc2-depleted cells (b) by dual-color IF for MCM (red; a–b) and PCNA (green; a′–b′). The arrow in (a) denotes an MCM-positive, PCNA-negative G1 cell (Prasanth et al, 2004). Total number of cells positive for MCM2 and MCM3 in Orc2-depleted cells was 77 and 76%, respectively, whereas the same for the control was 62 and 60%. In control cells, the MCM2+/PCNA+ was 24%, MCM2+/PCNA− 38%, MCM2−/PCNA+ 2% and MCM2−/PCNA− 36%. Similar results were obtained for MCM3 in control cells. In contrast, for Orc2 siRNA-treated cells, MCM2+/PCNA+ was 15%, MCM2+/PCNA− 62% and MCM2−/PCNA− 23%. Scale bar, 10 μm. RNAi-treated cells were assessed at 72 h.