Journal: iScience

Article Title: The basic domain of Suv39h2 buffers mitoxantrone-induced heterochromatin destabilization

doi: 10.1016/j.isci.2026.115626

Figure Lengend Snippet: Heterochromatin association of Suv39h2 is more resistant to mitoxantrone exposure than Suv39h1 or HP1α (A) Double-labeling immunofluorescence for Suv39h1-EGFP (left), Suv39h2-EGFP (right), and HP1α in D5-Suv39h1-EGFP and D5-Suv39h2-EGFP MEF cells exposed to a 1 h incubation with 0, 25, 50, and 100 μM mitoxantrone. Cells were labeled with α-GFP and α-HP1α antibodies and counterstained with DAPI. The percentages of cells with focal (white) or dispersed (yellow) fluorescence signals are indicated on the images. For each cell line and condition (i.e., mitoxantrone concentration), n ≥ 50 cells were analyzed. Scale bar is 5 μm. The chemical structure of mitoxantrone is shown on the right. (B) Immunofluorescence for H3K9me3 in D5-Suv39h1-EGFP and D5-Suv39h2-EGFP MEF cells exposed to mitoxantrone, as described in (A). (C) Mitoxantrone-mediated dispersion of Suv39h2-EGFP and H3K9me3 is reversible. D5-Suv39h2-EGFP MEF cells were incubated with mitoxantrone for 1 h and then cultivated in mitoxantrone-free medium. Samples were collected 2, 4, 6 (not shown), and 24 h after mitoxantrone removal and double-labeled for GFP and H3K9me3 and counterstained with DAPI. Scale bar is 5 μm. Quantification of the imaging data is shown in the line graph below. For each time point, n ≥ 60 cells were analyzed. (D) Time course for the mitoxantrone-mediated dispersion of Suv39h2-EGFP and H3K9me3. D5-Suv39h2-EGFP MEF cells were incubated with 100 μM mitoxantrone for 0, 30, 45, and 60 min. Cells were double-labeled for GFP and H3K9me3 and counterstained with DAPI. Scale bar is 5 μm. Quantification of the imaging data is shown in the line graph below. For each time point, n ≥ 65 cells were analyzed (3 independent experiments). Data are shown as mean ± SD. Asterisks indicate statistically significant differences ( p = 0.0001,∗∗∗, Šidak test).

Article Snippet: Samples were post-fixed in 4% PFA at room temperature for 15 min and mounted using VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories, H-1200-10).

Techniques: Labeling, Immunofluorescence, Incubation, Fluorescence, Concentration Assay, Dispersion, Imaging

Journal: iScience

Article Title: The basic domain of Suv39h2 buffers mitoxantrone-induced heterochromatin destabilization

doi: 10.1016/j.isci.2026.115626

Figure Lengend Snippet: Suv39h2 buffers heterochromatin integrity (A) Double-labeling immunofluorescence for Suv39h1-EGFP (left), Suv39h2-EGFP (right), and H3K9me3 in D5-Suv39h1-EGFP and D5-Suv39h2-EGFP MEF cells that were agarose-embedded, permeabilized, and incubated with 0.14, 0.4, 0.9, 1.1, and 2.0 M NaCl. Cells were labeled with α-GFP and α-H3K9me3 antibodies and counterstained with DAPI. Scale bar is 5 μm. (B) Violin plots show the quantification of mean fluorescence intensities of α-GFP (left) and α-H3K9me3 (right) signals. For each cell line and condition (i.e., NaCl concentration), n ≥ 30 cells were analyzed. Median values are indicated. Asterisks indicate statistically significant differences ( p = 0.0021, ∗∗, p < 0.00001, ∗∗∗∗, Mann-Whitney test). (C) Double-labeling immunofluorescence for Suv39h1-EGFP (left), Suv39h2-EGFP (right), and H3K9me3 in D5-Suv39h1-EGFP and D5-Suv39h2-EGFP MEF cells incubated with 0, 1.25, 2.5, and 5 μM cbl-0137. Cells were labeled with α-GFP and αH3K9me3 antibodies and counterstained with DAPI. Percentages of cells with focal (white) or dispersed (yellow) fluorescence signals are indicated on the images. For each cell line and condition (i.e., cbl-0137 concentration), n ≥ 60 cells were analyzed. Scale bar is 5 μm. The chemical structure of cbl-0137 is shown on the right.

Article Snippet: Samples were post-fixed in 4% PFA at room temperature for 15 min and mounted using VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories, H-1200-10).

Techniques: Labeling, Immunofluorescence, Incubation, Fluorescence, Concentration Assay, MANN-WHITNEY

Journal: iScience

Article Title: The basic domain of Suv39h2 buffers mitoxantrone-induced heterochromatin destabilization

doi: 10.1016/j.isci.2026.115626

Figure Lengend Snippet: The protective function of the basic domain can be transferred to Suv39h1 as an N-terminal fusion (A and B) Immunofluorescence of D5-Suv39h2ΔBD-EGFP (left) and D5-BD-Suv39h1-EGFP (right) MEF cells incubated with 0, 25, 50, and 100 μM mitoxantrone (A) or 0, 1.25, 2.5, and 5 μM cbl-0137 (B). Cells were double-labeled with α-GFP and α-HP1α or single-labeled with α-H3K9me3 antibodies and counterstained with DAPI. The percentages of cells with focal (white) or dispersed (yellow) fluorescence signals are indicated on the images. For each cell line and condition (i.e., mitoxantrone or cbl-0137 concentration), n ≥ 50 cells were analyzed. Scale bars are 5 μm. The chemical structures of mitoxantrone and cbl-0137 are shown on the right. (C) Heatmap shows the quantification of imaging data for GFP, HP1α, and H3K9me3 localization in D5-Suv39h1-EGFP, D5-BD-Suv39h1-EGFP, D5-Suv39h2-EGFP, and D5-Suv39h2ΔBD-EGFP MEF cells exposed to increasing concentrations of either mitoxantrone or cbl-0137. This heatmap summarizes the imaging analyses from A, 3B, C, A, 5B, and A. Percentages of cells with fluorescence signals over DAPI-dense foci are indicated by yellow (no overlap, dispersed) to blue (overlap, focal enrichment) gradient.

Article Snippet: Samples were post-fixed in 4% PFA at room temperature for 15 min and mounted using VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories, H-1200-10).

Techniques: Immunofluorescence, Incubation, Labeling, Fluorescence, Concentration Assay, Imaging