Figure 3. Altered localization of HP1-α,... | Rockefeller University Press

Figure 3.

$Figure 3. Altered localization of HP1-α, HP1-γ, and hMis12 proteins in Vpr-expressing cells. (A) HP1-α, HP1-β, and HP1-γ expression in DOX-treated MIT-23 and ΔVpr cells. Loading control (Cont): a major band of the chromosomal fraction corresponding to 15 kD by Coomassie brilliant blue staining. (B) Chronological changes in HP1-α and hRad21 in the chromosomal (Ch) versus whole-cell (Wh) fractions after DOX addition (days 0–3). βTub, β-tubulin; Vpr, Vpr expression. Data are representative of three independent experiments. (C) Cells were preextracted with PBS containing Triton X-100 and stained with anti–HP1-α antibody and Hoechst 33342. In the bottom right, the pictured cells are in interphase (top cell) and undergoing mitosis (bottom cell). Similar results were obtained in at least three independent experiments. (D) Chromosome spreads were immunostained for HP1 subtypes and CENP-H. Similar results were obtained in three independent experiments. (E) Mitotic cells were immunostained for hMis12 and CENP-A. (F) Chromosome spreads prepared from DOX-induced cells (48 h) were immunostained for hMis12 and CENP-A. (G, left) DOX-induced cells (48 h) were immunostained for hMis12 and CENP-A. (insets) Magnifications of the centromere. (right) The hMis12/CENP-A signal intensity ratio was measured during prophase and prometaphase. Gray bars, ΔVpr cells; black bars, MIT-23 cells. Values represent the mean ± SD (three experiments) of data generated using the cells shown on the left. *, P = 0.0008 versus Vpr-expressing cells in prophase. (H) Chromosome spreads immunostained with CENP-A and CENP-H antibodies and ACA antisera. Blue, DNA; red, CENP-A, ACA, or CENP-H. Vpr (−), DOX-treated ΔVpr cells at 48 h; Vpr (+), DOX-treated MIT-23 cells at 48 h. Bars: (C, E, and G) 10 µm; (D, F, and H) 5 µm; (G, insets) 0.5 µm.$

Altered localization of HP1-α, HP1-γ, and hMis12 proteins in Vpr-expressing cells. (A) HP1-α, HP1-β, and HP1-γ expression in DOX-treated MIT-23 and ΔVpr cells. Loading control (Cont): a major band of the chromosomal fraction corresponding to 15 kD by Coomassie brilliant blue staining. (B) Chronological changes in HP1-α and hRad21 in the chromosomal (Ch) versus whole-cell (Wh) fractions after DOX addition (days 0–3). βTub, β-tubulin; Vpr, Vpr expression. Data are representative of three independent experiments. (C) Cells were preextracted with PBS containing Triton X-100 and stained with anti–HP1-α antibody and Hoechst 33342. In the bottom right, the pictured cells are in interphase (top cell) and undergoing mitosis (bottom cell). Similar results were obtained in at least three independent experiments. (D) Chromosome spreads were immunostained for HP1 subtypes and CENP-H. Similar results were obtained in three independent experiments. (E) Mitotic cells were immunostained for hMis12 and CENP-A. (F) Chromosome spreads prepared from DOX-induced cells (48 h) were immunostained for hMis12 and CENP-A. (G, left) DOX-induced cells (48 h) were immunostained for hMis12 and CENP-A. (insets) Magnifications of the centromere. (right) The hMis12/CENP-A signal intensity ratio was measured during prophase and prometaphase. Gray bars, ΔVpr cells; black bars, MIT-23 cells. Values represent the mean ± SD (three experiments) of data generated using the cells shown on the left. *, P = 0.0008 versus Vpr-expressing cells in prophase. (H) Chromosome spreads immunostained with CENP-A and CENP-H antibodies and ACA antisera. Blue, DNA; red, CENP-A, ACA, or CENP-H. Vpr (−), DOX-treated ΔVpr cells at 48 h; Vpr (+), DOX-treated MIT-23 cells at 48 h. Bars: (C, E, and G) 10 µm; (D, F, and H) 5 µm; (G, insets) 0.5 µm.