Figure 3. CSD localization at centromeres is necessary and sufficient for HP1 to protect centromeric cohesion.

1. Lysates of asynchronous HeLa and HP1 DKO cells with or without stable expression of the indicated exogenous HP1α proteins were immunoblotted.
2. HeLa and the indicated stable cell lines were treated with nocodazole for 3 h. Mitotic chromosome spreads were immunostained. Scale bar, 10 μm.
3. HeLa and the indicated stable cell lines were exposed to MG132, then fixed at the indicated time points for DNA staining, and quantified in around 300 cells (n = 3).
4. HeLa and the indicated stable cell lines were exposed to MG132 for 8 h. Using mitotic chromosome spreads, the percentage of cells with cohesion loss was determined in around 100 cells.
5. HeLa and the indicated stable cell lines were treated with nocodazole for 3 h. Mitotic chromosome spreads were stained with CENP‐C antibodies and DAPI. The inter‐KT distance was measured on over 400 chromosomes in over 20 cells (unpaired t‐test).
6. HeLa and the indicated stable cell lines were analyzed for metaphase chromosome alignment in around 300 cells (n = 3) as in (C).
7. Lysates of asynchronous HeLa and HP1 DKO clone 2A4 cells stably expressing the indicated fusion proteins were immunoblotted.
8. The indicated stable cell lines were analyzed for metaphase chromosome alignment in around 300 cells (n = 3) as in (C).
9. The indicated stable cell lines were analyzed for cohesion loss in around 100 cells as in (D).
10. HeLa and the indicated stable cell lines were analyzed for the inter‐KT distance on over 400 chromosomes in over 20 cells (unpaired t‐test) as in (E).

Data information: Means and SDs are shown (C, E, F, H, J). See also Fig EV3.Source data are available online for this figure.