Fig. 3

Knockdown of HP1γ in germ cells results in mitotic abnormalities. a. Levels of P-S83-HP1γ in GC1 and GC2 cell lines. High levels of HP1γ and its phosphorylated Ser83 form are found in the cell lines, GC1 and GC2, by Western Blot. b. Knockdown of HP1γ in GC1 cells. shRNA-mediated knockdown of HP1γ results in reduction of HP1γ protein levels as shown by western blot. α-tubulin is used as loading control. c. Immunofluorescence of HP1γ knockdown in GC1 cells. Immunofluorescence of control cells (shCTRL) shows colocalization of P-S83-HP1γ staining in green and γ-tubulin in red, creating a yellow signal in the overlay. DNA is counterstained with DAPI (blue). ShRNA knockdown of HP1γ abolishes P-S83-HP1γ staining (loss of green signal) and results in centrosomal abnormalities compared with control cells. A representative shHP1γ cell is shown where centrosomes are labeled by γ-tubulin staining (red) to demonstrate aberrant spindle pole number and localization during mitosis. d. Quantification of mitotic abnormalities. Quantification reveals a significantly high rate of centrosomal abnormalities in the shHP1γ cells vs shCTRL, 26.5 % and 1.5 % respectively. e. HP1γ knockdown in GC1 cells results in decreased cell division. Mitotic index assay confirms that shHP1γ cells have decreased cell division compared to shCTRL cells (78.1 % ± 1.3 %; normalized to shCTRL), likely as a result of these mitotic abnormalities