Fig. 5: Segmentation-based SG shape analysis.

a ISM reconstructed lifetime-based image of SK-N-BE WT cells expressing G3BP1-eGFP under oxidative stress before the RNAseA treatment. Bottom-right zoomed region in the white box. Scale bar 10 μm. b Phasor-based segmentation. ISM intensity images were obtained by back-projecting the points with a long lifetime (magenta) and a short lifetime (yellow). Scale bar 10 μm. c Outlines of the segmented SGs in the zoomed region. The numbers correspond to the circularity. d ISM reconstructed lifetime-based image of SK-N-BE WT cells expressing G3BP1-eGFP under oxidative stress during the RNAseA treatment. Bottom-right zoomed region in the white box. Scale bar 10 μm. e Phasor-based segmentation. ISM intensity images were obtained by back-projecting the points with a long (magenta) and a short lifetime (yellow). Scale bar 10 μm. f Outlines of the segmented SGs in the zoomed region. The numbers correspond to the circularity. g Box-plots of the circularity of the segmented SGs in SK-N-BE WT cells (left) and in mutated cells (right) before the addition of RNAseA and during the RNase treatment. The data are from two independent experiments (WT: n = 106 before RNAseA and n = 72 during RNAseA; mutated: n = 51 before RNAseA and n = 62 during RNAseA). The horizontal line in each box represents the median, the edges are the 25th and 75th percentile, and the vertical line extends to the minimum and maximum data points. h Box-plots of the area of the segmented SGs in SK-N-BE WT cells (left) and in mutated cells (right) before the addition of RNaseA and during the RNase treatment. The data are from two independent experiments (WT: n = 106 before RNAseA and n = 72 during RNAseA; mutated: n = 51 before RNAseA and n = 62 during RNAseA). The horizontal line in each box represents the median, and the edges are the 25th and 75th percentile. The single points are the outliers. i Confinement strength in relation to the diffusion coefficient of G3BP1 (diamonds) during the RNaseA treatment in WT SK-N-BE cells overlaid to the clustered data (empty squared). The color depicts the fluorescence lifetime. j Confinement strength in relation to the diffusion coefficient of G3BP1 (diamonds) protein during the RNaseA treatment in mutated SK-N-BE cells overlaid to the clustered data (empty squared). The color depicts the fluorescence lifetime value. Source data are provided as a Source Data file.