dna counterstaining  (Santa Cruz Biotechnology)

Journal: The FASEB Journal

Article Title: Polyalanine Expansion in PABPN1 Alters the Structure and Dynamics of Its Nuclear Aggregates in Differentiated Muscle Cells

doi: 10.1096/fj.202501097R

Figure Lengend Snippet: Morphological analysis of nuclear PABPN1 puncta between proliferating and differentiated myonuclei. (A–E) Experiments in proliferation conditions 72 h Dox‐treatment; (F–G) Experiments in differntion conditions 72 h Dox‐treatment. (A) Representative confocal images of Ala10‐YFP and Ala16‐YFP (in green) in a single myonucleus (DAPI counterstain is in blue) and masks of YFP puncta at low and high threshold (TH25 and TH400, respectively). The scale bar is 10 μm. (B) Boxplot showing the average number of YFP puncta per nucleus at low and high thresholds. N = 25 nuclei. (C–E) Analysis was performed in TH25. (C, D) Box plots show the mean and intranuclear variation of puncta area or circularity. Circles represent single nuclei. (E) A linear regression analysis of puncta [Log2 (area and circularity)], each point represents a single punctum. The significance of the F‐test, the slope (S) and the coefficient (R) are indicated. (F) Representative refractive index images of myotubes overlaid with fluorescence signal. The scale bar is 20 μm. (G) Box plots of mean YFP intensity (left), puncta area (middle), and circularity (right). Statistical significance was determined by one‐way Anova. p < 0.05, 0.005, 0.001 are indicated by *, ***, ****, respectively.

Article Snippet: In living cells, nuclear counterstain was made with Sir700‐DNA kit (Spirochrome # SC015).

Techniques: Refractive Index, Fluorescence

Journal: The FASEB Journal

Article Title: Polyalanine Expansion in PABPN1 Alters the Structure and Dynamics of Its Nuclear Aggregates in Differentiated Muscle Cells

doi: 10.1096/fj.202501097R

Figure Lengend Snippet: Kinetics of Ala10 and Ala16 puncta accumulation. (A) Representative confocal images of Ala10 and Ala16 cell cultures treated with Dox for 0, 12, and 72 h. Cell cultures were immunostained with anti‐MyHC, nuclei counterstained with DAPI, YFP shown in green. The scale bar is 20 μm. (B) YFP puncta MFI accumulation over time in multinucleated MyHC‐positive cells (right angle and solid line) and mononucleated cells (circle and dashed line). Mean and standard deviation are based on 100 nuclei. (C) Box plot of the average number of puncta per nucleus in multinucleated cells after 36 h of Dox treatment. Each dot represents one multinucleated cell. Mean and standard deviation are from 15 multinucleated cells. (D–F) Imaging in living cells. Nuclear counterstain was made with Sir‐700. (D) Representative confocal images of Ala16 differentiated cell cultures after 12 and 72 h of Dox treatment. Multinucleated and mononucleated objects are outlined with solid or dashed lines, respectively. Scale bar is 50 μm. (E, F) Quantification of Ala16‐YFP puncta area (E) and circularity (F) in multinucleated or mononucleated objects, denoted by continuous or dashed lines, respectively. Mean and standard deviation are from single nuclei: N = 41–73 mononucleated objects and N = 63–137 multinucleated objects. (G, H) Imaging was carried out in proliferating cell cultures starting 16 h after Dox treatment. (G) Overlay image of three frames (2‐s interval) from time‐lapse imaging. Each time frame is represented by a color (red, green, or blue); white indicates overlap of puncta. The magnification of the boxed area and the segmented white area (cyan) are shown in the right panels. Scale bar is 10 μm. (H) Dot plot of the ratio between the white area to the colored area in Ala10 and Ala16 nuclei. Standard deviation is from N = 10 nuclei from two experiments. Statistical significance was assessed by one‐way ANOVA. p < 0.005, 0.001 are indicated by **, ***, ****, respectively.

Article Snippet: In living cells, nuclear counterstain was made with Sir700‐DNA kit (Spirochrome # SC015).

Techniques: Standard Deviation, Imaging