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Fig. 5
Figure 5. Solitary disseminated tumor cells have low srGAP1 expression (A) Tissue staining of MDA-MB-231 single tumor cells and clusters in Ctrl mice lungs. Yellow arrow points to single cell. Top: GFP-labeled tumor cell, DAPI for nuclei. Bottom: srGAP1 staining. Scale bar, 10 μm. (B) srGAP1 intensity of MDA-MB-231 tumor cells and clusters in Ctrl mice lung (mean intensity in a defined area calculated for individual tumor cells and clusters). Data pooled over two Ctrl mice, three 450 × 450 μm sections quantified per mouse. Mann Whitney U test, two-tailed. Scale bar, 10 μm. Data represented as a violin plot; horizontal lines are median and interquartile range. (C) Top: time-lapse imaging of MDA-MB-231 Ctrl and srGAP1 KD #1 cells expressing H2B-RFP and a DHB:Venus sensor for Cdk2 activity. Frames from live-cell imaging of cells plated on fibrillar collagen, time in hours. Scale bar, 10 μm. Bottom: schematic representing cells expressing DHB:Venus sensor: nuclear expression is G0/G1, nuclear and cytoplasmic expression is S, and cytoplasmic expression is G2/M. (D) Quantification of cytoplasmic to nuclear (C/N) ratio of DHB:Venus sensor for Cdk2 activity over time for MDA-MB-231 Ctrl and srGAP1 KD #1 cells from (C). n = 24 (Ctrl), 34 (srGAP1 KD #1) cells, two independent experiments. C/N ratio calculated for five frames (2 h). Data represented as mean ± SEM. (E) Average cytoplasmic to nuclear ratio of MDA-MB-231 Ctrl and KD #1 cells expressing the DHB:Venus sensor for Cdk2 activity and H2B-RFP. C/N calculated from live-cell imaging (averaged over 2 h) of cells plated on fibrillar collagen. n = 24 (Ctrl), 34 (srGAP1 KD #1) cells, two independent experiments. Unpaired t test, two-tailed. (F) Images of lungs from Ctrl and srGAP1 KD #1 MDA-MB-231 xenografts expressing H2B-RFP and a DHB:Venus sensor for Cdk2 activity. Scale bars, 50 μm (left), 10 μm (right). (G) Number of MDA-MB-231 cells in the lung with nuclear DHB:Venus sensor expression relative to lesion area. Ten fields of view (255 × 255 μm) per mouse, data pooled over one Ctrl and one srGAP1 KD #1 mouse, each point is a field. Mann-Whitney U test, two-tailed. Data represented as a box and whisker plot, with median and interquartile range in the box and min and max as whiskers.