Fig. 4. Acetylation impairs nuclear condensation of HP1γ in vivo and vitro.

a Upper: representative images and quantification of puncta count for endogenous foci of HP1γ in WT and BR MM.1 S and LP-1 cells. Error bars represent S.E.M., n  =  3 (biologically independent experiments). Lower: quantification of puncta count is shown in images. b FRAP assay (upper) and kinetic recovery times (lower) for a HP1γ-GFP focus by 488 nm laser for 5″bleaching and 40″recovery in HEK293T cells transfected with HDAC1 or vector control (mean ± s.d.; n =  3 independent experiments). c Upper: Representative images for HP1γ foci in Vector and HDAC1 overexpression MM cells (n = 3). Lower: Quantification of puncta count is shown in images. d Visualization of turbidity associated with droplet formation in vitro for the GFP-vector control (Ctrl), (HP1γ-IDR1)-WT, -K5Q, K5R or -KallQ. e Turbidity (OD600) of WT, K5Q, K5R, and KallQ mutants of HP1γ-IDR1 in 200 mM NaCl and 10% PEG was measured (mean ± s.d.; n =  3 independent experiments). f FRAP assay for the recovery of fluorescence intensity of WT-, K5R- and K5Q-HP1γ-FL in HEK293T cells (mean ± s.d.; n =  3 independent experiments). g Microscopy images of GFP fusion proteins of WT-, K5Q- and K5R-HP1γ-FL before and after partial droplet photobleaching in vitro (mean ± s.d.; n =  3 independent experiments). h Visualization of turbidity associated with droplet formation in vitro for mock reaction and deacetylated HP1γ protein. i Turbidity (OD600) of mock reaction and deacetylated HP1γ protein in 200 mM NaCl and 10% PEG was measured (mean ± s.d.; n =  3 independent experiments). j Quantitative assessment of HR activity in the HEK293T cells expressing vector control or K5R-HP1γ via flow cytometry assay for the percentage of GFP⁺ cells among RFP⁺ cells (mean ± s.d.; n =  3 independent experiments). P values were determined by one-way ANOVA (b, i), two-way test (e, f, g) and Student’s t test (a, c, j). Source data are provided as a Source data file.