P-Ser83-HP1γ is necessary for proper mitotic function. (A) Stable knockdown of HP1γ in HeLa cells. Western blot of HP1γ levels (top) is shown from HeLa cell lysates to confirm stable lentiviral-mediated shHP1γ compared to shCTRL. α-tubulin serves as a loading control (bottom). (B) HP1γ knockdown eliminates P-Ser83-HP1γ at the spindle poles. Representative images are shown for immunofluorescence on shCTRL and shHP1γ HeLa cells to demonstrate specific loss of P-Ser83-HP1γ (green) staining. Co-staining with γ-tubulin (red) was performed to establish the localization of the spindle poles. Cells were counterstained with DAPI and the overlay is shown. Scale bar represents 5 μM. (C) Mitotic aberrations caused by HP1γ knockdown are rescued by wild type, but not S83A-HP1γ mutant. Mitotic aberrations were quantified for shCTRL and shHP1γ cells. In order to determine if Ser83 phosphorylation plays a role in this function, shHP1γ cells were infected with adenovirus carrying wild type or S83A-HP1γ mutant. While reintroduction of wild type HP1γ was able to significantly rescue this effect, S83A-HP1γ mutant was not, implicating Aurora A-mediated phosphorylation in this phenomenon. For each condition, 200 mitotic cells were analyzed. Western blot is shown of endogenous HP1γ levels (inlay, top) as well as transduced His-tagged wild type and S83A-HP1γ mutant proteins (arrow). α-tubulin serves as a loading control (inlay, bottom). *Transduction with EV control did not change the number of abnormalities observed with shHP1γ. (D) Mitotic aberrations observed in stable shHP1γ cells include multipolar spindles, centrosome disruption and lagging, unorganized chromosomes. Representative images are shown for the types of observed mitotic aberrations. γ-tubulin (red) marks spindle poles with DAPI counterstain to show condensed mitotic chromosomes. Scale bar represents 5 μM. DAPI, 4',6-diamidino-2-phenylindole; EV, empty vector; P-Ser83-HP1γ, phosphorylation of HP1γ at serine 83; Ser83, serine 83; shCTRL, shRNA control; shHP1γ, shRNA knockdown of HP1γ; shRNA, short hairpin RNA.