Levels of P-Ser83-HP1γ are cell cycle-dependent, increasing significantly in G
/M. (A) Inhibition of HP1γ phosphorylation in vivo by the cell cycle inhibitor, roscovitine. HeLa cells incubated with roscovitine, an inhibitor of cell cycle progression at the G1/S and G2/M checkpoints, display a dose-dependent inhibition of phosphorylation as shown by anti-P-Ser83-HP1γ (top). α-tubulin is shown as a loading control (bottom). (B) P-Ser83-HP1γ levels are high in mitotic arrested cells. Cell extracts were obtained from a normal cycling population (con), cells treated with aphidicolin (aph) to arrest cells in G1/S phase (G1/S), or mitotic-arrested cells (G2/M) from treatment with nocodazole (noc). An increase of P-Ser83-HP1γ levels in mitosis is shown by comparison of anti-P-Ser83-HP1γ (top) with total HP1γ (bottom). (C) P-Ser83-HP1γ levels through the cell cycle. HeLa cells were synchronized by double thymidine block and cell extracts were obtained at subsequent time points of release. P-Ser83-HP1γ levels are highest approximately 8 to 10 hours post-release, which corresponds to an increase in the presence of other mitotic markers, including P-Ser10-H3, Aurora A and Aurora B, indicating M phase entry. The relative intensity indicated below was calculated as P-Ser83-HP1γ/pan-HP1γ ratios and normalization with the ratio of 0 hour. aph, aphidicolin; con, control; noc, nocodazole; P-Ser10-H3, phosphorylation of histone H3 at serine 10; P-Ser83-HP1γ, phosphorylation of HP1γ at serine 83.