Fig 2: Both CBX8 and PTEN suppress H3K27Me3 in mitosis. (A) HeLa cells were synchronized by sequential treatment with thymidine and nocodazole as described in Experimental Procedures. Mitotically arrested cells were then released into the cell cycle for various times as indicated. Equal amounts of cell lysates were blotted for H3K27Me3, H2AK119Ub1, H3 and H2A, as well as a panel of cell cycle components as indicated. (B) HeLa cells transfected with CBX8 siRNAs, PTEN siRNAs or control siRNAs for 24 h followed by nocodazole treatment for 16 h. Equal amounts of cell lysates of various treatments were blotted for PTEN, CBX8, H3K27Me3, H2AK119Ub1, cyclin B1, phosphorylated H2Ax (p‐H2Ax) and β‐actin

Fig 3: Chromobox Homolog protein 8 is involved in regulating mitosis. (A) HeLa cells seeded in triplicates were transfected with CBX8 siRNAs or control siRNAs for various times. Transfected cells were then subjected to cell proliferation assays as described in Experimental Procedures. Experiments were repeated for three times. Standard error bars are shown. Stars (** and ***) denote statistically significant between treatment groups (**P < .001 and ***P < .0001 compared to control cells). (B) HEK293T cells were transfected with an expression plasmid coding for FLAG‐CBX8 or with empty vector (EV) for 24 h followed by treatment with nocodazole for 16 h. Both asynchronized and mitotic cells were collected and lysed. Equal amounts of cell lysates were blotted with antibodies to FLAG (CBX8), cyclin B1, PTEN, p‐H3, Eg5, H3K27Me3, H2AK119Ub1 and β‐actin

Fig 4: A schematic model that depicts regulation of chromatin modifications by PTEN and CBX8 during mitotic progression. PTEN and phosphorylated CBX8 interact with MCC and negatively regulates trimethylation of histone H3K27 during mitosis. Reduced levels of H3K27Me3 are necessary for mitotic progression and exit. H3K27Me3 levels are high in G1

Fig 5: Phosphatase and tensin homolog regulates MCC and mitotic progression. (A) HeLa cells were transfected with PTEN‐specific siRNAs or control siRNAs for 24 h and then treated with nocodazole (Noc) for 16 h, after which mitotic cells were collected by shake‐off. After washing, mitotic cells were released into the cell cycle. At various times post‐release, cells were lysed and equal amounts of cell lysates were blotted for PTEN and MCC components including BubR1, Cdc20, Mad2 and Bub3. Modified (sumoylated or phosphorylated) forms of BubR1 are indicated. HeLa cell line was used here because it has been widely recognized for studying cell cycle regulation, largely because of ease of collecting mitotic cells via shake‐off. (B) HEK293T cells were transfected for 24 h with plasmid constructs expressing FLAG‐tagged CBX2, CBX4, CBX6, CBX7 or CBX8. After transfection, cell lysates were immunoprecipitated with the anti‐FLAG antibody. FLAG immunoprecipitants, along with lysate inputs, were blotted with the anti‐FLAG antibody and antibodies to BubR1, Cdc20, Mad2 and PTEN. HEK293T cells were used here because these cells are amenable to transfection. (C) HeLa cells were treated with nocodazole for 16 h, after which mitotic cells were collected by shake‐off. Cell lysates of asynchronized (AS) cells and mitotic cells (rounded up, R. up) were immunoprecipitated with the anti‐CBX8 antibody or with control IgG. CBX8 Immunoprecipitants, along with lysate inputs, were blotted for CBX8, BubR1 and PTEN