Figure 2. Monitoring H3K9ac levels in living cells.

(a) H3K9ac-mintbody as a measure of H3K9ac levels. The more H3K9ac levels increase, the more mintbodies bind to chromatin, resulting in more nuclear accumulation. (b) FRAP. A spot (yellow circle) was bleached in cells expressing H3K9ac-mintbody v3. FRAP curves were fitted to a reaction-diffusion model²³ to obtain t_on (s), t_off (s), and the bound/free fraction. Averages with s.e.m. are shown (TSA, n = 19; DMSO, n = 18). (c) Nuclear concentration of H3K9ac-mintbody. Cells expressing H3K9ac-mintbody v3 were incubated in presence or absence of TSA, and typical confocal images are shown on the left. Nuclear to cytoplasmic intensity ratio was measured and plotted (average with s.e.m.). The numbers of analyzed cells were 31 (vehicle), 31 (1 nM), 30 (10 nM), and 40 (100 nM). (d) After 8 hr incubation in 100 nM TSA, cells were washed and incubated in normal medium, Nuclear to cytoplasmic intensity ratio was measured and plotted (average with s.e.m; n = 32). Scale bars, 10 μm.