Journal: iScience
Article Title: Single-molecule imaging quantifies oncogenic KRAS dynamics for enhanced accuracy of therapeutic efficacy assessment
doi: 10.1016/j.isci.2025.113374
Figure Lengend Snippet: PS depletion attenuates the transient trapping of KRAS WT and oncogenic mutant molecules (A; left) Schematic representation of single-molecule imaging of a PS probe (evectin2 [2xPH]) in SW48 cells, with (bottom) or without (top) PS depletion via PSD expression. (Right) Schematic representation of single-molecule imaging of KRAS in SW48 cells with (bottom) or without (top) PS depletion. Activated KRAS (KRAS-GTP) forms nanoclusters facilitated by PS and/or CRD of BRAF, which associates with PS in the membrane. (B) Fluorescence images of SF650B-Halo7-evectin2 (2xPH) and mCherry-PSD in the presence (bottom) or absence (top) of PSD expression. Images were acquired using oblique angle illumination and TIRFM. Single fluorescent spots of evectin2 (2xPH) recruited to the PM are indicated by yellow arrowheads. (C) Quantification of evectin2 (2xPH) fluorescent spots recruited to the PM with or without PSD expression. Values were normalized to both total probe expression (measured via whole-cell fluorescence under oblique-angle illumination) and the observation area. (D–F) Temporal fractions of transient trapping (D), distributions of trapping durations (E), and trapping zone sizes (F) for KRAS WT, G13D, and G12V, with or without PSD expression, measured 2−5 min after EGF stimulation. The normalized number of recruited PS probe spots and the temporal fraction of trapped molecules are presented as box-and-whisker plots, displaying the minimum, maximum, sample median, sample mean (circle), first and third quartiles, and whiskers extending to a maximum of 1.5× interquartile range beyond the box. The size distribution of individual trapping zones is presented using both violin plots and box-and-whisker plots, illustrating the sample median, sample mean (circle), first and third quartiles, and whiskers extending to a maximum of 1.5× interquartile range beyond the box. Statistical analysis was performed using Welch’s t test.
Article Snippet: For covalent labeling of HaloTag7 with SF650B, cells were incubated with 50 nM SF650B-Halo ligand (GORYO CHEMICAL) in culture medium for 30 min at 37°C, followed by three washes with fresh medium to eliminate unbound ligand.
Techniques: Mutagenesis, Imaging, Expressing, Membrane, Fluorescence, Whisker Assay
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![PS depletion attenuates the transient trapping of KRAS WT and oncogenic mutant molecules (A; left) Schematic representation of single-molecule imaging of a PS probe (evectin2 [2xPH]) in SW48 cells, with (bottom) or without (top) PS depletion via PSD expression. (Right) Schematic representation of single-molecule imaging of KRAS in SW48 cells with (bottom) or without (top) PS depletion. Activated KRAS (KRAS-GTP) forms nanoclusters facilitated by PS and/or CRD of BRAF, which associates with PS in the membrane. (B) Fluorescence images of <t>SF650B-Halo7-evectin2</t> (2xPH) and mCherry-PSD in the presence (bottom) or absence (top) of PSD expression. Images were acquired using oblique angle illumination and TIRFM. Single fluorescent spots of evectin2 (2xPH) recruited to the PM are indicated by yellow arrowheads. (C) Quantification of evectin2 (2xPH) fluorescent spots recruited to the PM with or without PSD expression. Values were normalized to both total probe expression (measured via whole-cell fluorescence under oblique-angle illumination) and the observation area. (D–F) Temporal fractions of transient trapping (D), distributions of trapping durations (E), and trapping zone sizes (F) for KRAS WT, G13D, and G12V, with or without PSD expression, measured 2−5 min after EGF stimulation. The normalized number of recruited PS probe spots and the temporal fraction of trapped molecules are presented as box-and-whisker plots, displaying the minimum, maximum, sample median, sample mean (circle), first and third quartiles, and whiskers extending to a maximum of 1.5× interquartile range beyond the box. The size distribution of individual trapping zones is presented using both violin plots and box-and-whisker plots, illustrating the sample median, sample mean (circle), first and third quartiles, and whiskers extending to a maximum of 1.5× interquartile range beyond the box. Statistical analysis was performed using Welch’s t test.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_4429/pmc12424429/pmc12424429__gr4.jpg)