sir dna  (Cytoskeleton Inc)

Journal: iScience

Article Title: The TRIP12’s intrinsically disordered region induces chromatin condensates and interferes with nuclear processes

doi: 10.1016/j.isci.2025.114592

Figure Lengend Snippet: The formation of TRIP12 induced-chromatin condensates is a dynamic and reversible mechanism driven by PPPS (A) Visualization of CREST (top) and LAMIN B1 (bottom) and chromatin condensates in TRIP12-IDR-expressing HeLa S3 cells by immunofluorescence. Nuclei were counterstained with DAPI. Scale bars represent 2 μm. (B) Representative images of chromatin condensate formation and GFP expression in IDR-GFP expressing HeLa S3 cells by live cell microscopy. Images were acquired every hour for 24 h after transfection. For clarity, representative image of a cell 8 h after transfection and every 2 h are represented. Nuclei were counterstained with SiR-DNA-647 nm. Scale bars represent 5 μm. (C) Quantification of GFP expression and DNA granularity in IDR-GFP-expressing HeLa S3 cells obtained by live cell microscopy. Images were acquired every hour for 24 h by live cell confocal microscopy. GFP intensity and DNA granularity were determined using Columbus and Fiji software. Results are expressed as mean ± SEM of at least five independent cells. A Spearman r coefficient test and a two tailed-p value are indicated. Error bars represent the standard deviation of the mean. (D) Expression of IDR-GFP, FLAG-tagged anti-GFP degrader in VHL-NbGFP4-FLAG expressing HeLa S3 cells determined by western blot 24 h after increasing doses of doxycycline (0.25, 0.5, and 1 μg/mL). The immunoblots are representative of three independent experiments. HSP90 protein level was used as a loading control. (E) Representative images of VHL-NbGFP4-FLAG expressing in HeLa S3 cells transfected with IDR-GFP construct in the presence or not of doxycycline (1 μg/mL) for 24 h obtained by immunofluorescence. Nuclei were counterstained with DAPI. Scale bars represent 10 μm. (F) Determination of DNA granularity relative to IDR-GFP expression level in VHL-NbGFP4-FLAG expressing in HeLa cells transfected with IDR-GFP construct in the presence of doxycycline (1 μg/mL) for 24 h or vehicle. For each cell, DAPI granularity and GFP expression were determined as described in “ ” on more than 50 cells. Blue and green filled circles correspond to individual non-transfected and transfected cells, respectively. The linear regression curve is indicated in black. A Spearman r coefficient test and a two tailed-p value are indicated (G) Representative images of DNA condensates and IDR-GFP expression in VHL-NbGFP4-FLAG expressing HeLa S3 cells transfected with IDR-GFP construct every hour (for 13 h) visualized by cell live microscopy after addition of doxycycline (1 μg/mL). DNA is stained with SiR-DNA-647 nm. Scale bars represent 2 μm. (H) Quantification of DNA granularity and GFP intensity in VHL-NbGFP4-FLAG expressing HeLa S3 cells transfected with IDR-GFP construct every hour (for 13 h) visualized by cell live microscopy after addition of doxycycline (1 μg/mL). Results are expressed ±SEM of at least five independent cells. A Spearman r coefficient test and a two tailed-p value are indicated. Error bars represent the standard deviation of the mean. (I) Representative images of DAPI organization in IDR-GFP expressing HeLa S3 cells in the presence of 1,6-hexanediol 0.5% or vehicle for 18 h. Cells with three different GFP intensities are represented. Nuclei were counterstained by DAPI. (J) Determination of DAPI granularity in IDR-GFP expressing HeLa S3 cells in the presence of 1,6-hexanediol 0.5% or vehicle for 18 h as described in “ .” Error bars represent the standard deviation of the mean. (K) IDR-GFP mobility in DNA condensates by FRAP analysis. H2B-dsRed expressing HeLa S3 cells were transfected with IDR-GFP construct or H2B-GFP used as control. After 24 h, GFP fluorescence was photo-bleached using an FRAP module of confocal microscope. The recovery of fluorescence was measured every second for 35 s. Representative images of IDR-GFP and H2B-GFP expressing cells with bleached and non-bleached areas at indicated times are represented (left). The graph represents the mean ± SD of GFP fluorescence intensity obtained from four independent cells. (L) IDR-GFP mobility in DNA condensates by half-bleached FRAP analysis. HeLa S3 cells were transfected with IDR-GFP construct. After 24 h, GFP fluorescence in a half of a condensate was photo-bleached using an FRAP module of confocal microscope. The recovery of GFP fluorescence was measured every 0.5 s for 2 min. Representative images of an IDR-GFP expressing cell with bleached and non-bleached areas before and just after bleaching are represented (left). Recovery curves of the bleached half (green) and non-bleached half (magenta) are shown in the right figure, n = 15. Data are mean ± SEM. Scale bars represent 2 μm.

Article Snippet: SiR-DNA , Spirochrome , Cat# SC007.

Techniques: Expressing, Immunofluorescence, Microscopy, Transfection, Confocal Microscopy, Software, Two Tailed Test, Standard Deviation, Western Blot, Control, Construct, Staining, Fluorescence

Journal: Genes & Development

Article Title: SOX2 phosphorylation during mitosis limits genomic damage

doi: 10.1101/gad.352664.125

Figure Lengend Snippet: Unconstrained mitotic SOX2 activity causes mitotic errors and genomic instability. ( A ) Volcano plot of bulk RNA-seq comparing 3A-SOX2 with WT-SOX2 after 72 h of 1 µg/mL doxycycline (DOX,) induction ( n = 3 replicates). Dashed lines mark P -value = 0.05 and log 2 fold change = 1. ( B ) Gene set enrichment analysis (DNA repair signatures) of the ranked gene list shown in A . ( C ) Time-lapse confocal images of WT-SOX2-GFP and 3A-SOX2-GFP mNSCs stained with SiRDNA (magenta) during mitosis; minutes after nuclear envelope breakdown are indicated. Scale bar, 10 µm. ( D ) Scatter plots (median ± IQR) of the time spent in prometaphase and telophase for WT versus 3A cells (15–20 mitoses; three experiments). ( E , left ) Representative DAPI images of a normal mitosis, lagging chromatin/anaphase bridge, and chromosome compaction defect. ( Right ) Stacked bar chart of event frequencies in parental BL6, WT-SOX2, and 3A-SOX2 lines (≥100 mitoses; three experiments). ( F ) Immunoblots of whole-cell lysates collected 0, 24, and 72 h after DOX induction ( top ) and probed for SOX2, GAPDH, and γH2AX. ( G ) Immunofluorescence for γH2AX (red) in BL6, WT-SOX2, and 3A-SOX2 cells with (DOX + ) or without (DOX − ) induction. Nuclei were stained with DAPI (blue) and SOX2-GFP (green). Scale bar, 10 µm. ( H ) Box and whisker plot quantifying normalized γH2AX intensity per nucleus from images in G (≥150 nuclei; three experiments). ( I ) Immunoblots of BL6 and FLAG-WT-SOX2 or FLAG-3A-SOX2 lines of cells synchronized with 10 µM RO3306 and then released for 0, 45, or 90 min. The dashed line represents where lanes were omitted. Blots were probed for SOX2, phospho-histone H3 (Ser10), γH2AX, and GAPDH.

Article Snippet: Nuclei were counterstained with 1 μg/mL 4′,6′-diamidino-2-phenylindole (DAPI) or SiRDNA (Spirochrome) for 5 min.

Techniques: Activity Assay, RNA Sequencing, Staining, Western Blot, Immunofluorescence, Whisker Assay