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: Phosphorylated CBX8 interacts with chromatin PTEN. (A) Schematic presentations showing various deletion and mutation constructs of PTEN. (B) HEK293T cells were co‐transfected plasmids expressing FLAG‐CBX8 and GST‐PTEN, or GST‐tagged individual PTEN deletion mutants as indicated, for 24 h. Equal amounts of cell lysates were immunoprecipitated with the FLAG antibody. Immunoprecipitants, along with lysate inputs, were blotted with the antibody against GST (for PTEN or its mutants) and FLAG. (C) A proposed model depicting how CBX8 mediates the physical interaction with PTEN. (D) HeLa cells were treated with nocodazole or vehicle for 16 h. Mitotic cells (Rounded‐up) and asynchronized cells (AS) were collected and fractionated into the cytoplasmic (for asynchronized cells), nucleoplasmic (for mitotic cells) (Cyto/Solu) and chromatin (Chro) parts. Equal amounts of whole cell lysates (WCL) and fractionated protein lysates were blotted for CBX8, PTEN, BubR1, PARP‐1 and α‐Tubulin. (E) HeLa cells seeded on chamber slides were fixed and processed for staining with anti‐PTEN (Green channel) and anti‐CBX8 (Red channel) antibodies. DNA was stained with DAPI (Blue channel). Stained cells were examined under a confocal microscope. Representative images are shown (scale bar = 25 μm)

Fig 4: PRC1 subunits localize to restricted OE subpopulations. (A) PRC1 complexes are highly diverse, owing to the multiple variants that exist for each subunit. For instance, a canonical PRC1 contains one of five possible CBX variants and one of six known PCGF variants. (B) Gene expression assays by RT–qPCR from expansion–competent basal cell cultures reveal heterogeneous expression of multiple PRC1 subunits. The PRC2 gene Ezh2, expressed in basal cells, is included for comparison. Gene expression is normalized to RPO, n=3 replicates, bars=SD. (C, D) The PCGF proteins BMI1 (PCGF4) and MEL18 (PCGF2) have complementary expression patterns in adult OE tissue. MEL18 (C) is widely present in globose basal cells (BCs) and immature neurons (iOSN), as well as weakly expressed in the apical sustentacular and microvillar cell layers (Sus/mV); BMI1 (D) is present in mature neurons (mOSN) as well as occasional upstream globose cells (arrows), previously reported to be the SOX2+ subset; note the absence of BMI1 label in the thin iOSN region in D, in contrast to MEL18 label in C. (E, F) CBX8 is a neuron–specific PRC1 subunit in the OE; CBX8 is restricted to the neuronal layers by immunohistochemistry (E); Western blot (F) confirms antigen of correct size is detected by anti–CBX8 from OE extracts. Nuclei are stained with DAPI (blue) C–E, Bar=10μm in C, 50μm in E.

Fig 5: Chromobox Homolog protein 8 is phosphorylated during mitosis. (A) HeLa cells were synchronized with thymidine (Thy) and nocodazole (Noc) treatments, after which mitotic cells were collected by shake‐off. Mitotic cell lysates were treated with or without λ‐phosphatase at room temperature for 1 h and then blotted for CBX8 and vinculin. (B) HeLa cells were synchronized by treatment with thymidine followed by nocodazole. Mitotic cells were washed and released into the cell cycle for various times as indicated. Cell lysates were blotted for CBX8, BubR1, cyclin B1, phosphorylated H3 (p‐H3), cyclin A2, cyclin D3 and β‐actin