Fig. 3. At high hypertonic load, the nuclear periphery re-organizes: (A) Co-staining of chromatin (red, ToPro3 in MCF7 and H2B-GFP in HeLa) and anti-LaminAC (green) in MCF7 (top row) and HeLa cells (bottom row). The lamin-rim stain allows us to demonstrate retraction of peripheral chromatin from the nuclear envelope, which occurred in addition to the compaction of chromatin upon incubation with 320 mM sucrose load, but not with 160 mM sucrose. (B) Co-staining of chromatin (red, ToPro3) with anti-SC35 (green) in MCF7 cells. Together with the retraction of chromatin in 320 mM sucrose medium, SC35-positive foci established in the outer border of peripheral chromatin. These peripheral speckles were smaller in size than those of the inner nucleus (top, left). In control cells (top, right) and cells after incubation in 160 mM sucrose (top, middle), peripheral speckles were never observed. Incubation with sorbitol (bottom, left) or NaCl (bottom, middle) to achieve osmotic loads equivalent to 320 mM sucrose (see Table 1) also induced chromatin compaction and formation of peripheral speckles. By contrast, 10 kDa dextran added in a weight/concentration comparable to 320 mM sucrose had no noticeable effect (bottom, right). Substances were added to the normal growth medium (DMEM) as indicated and cells incubated for 20 minutes prior to fixation. Bars, 10 µm.