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.
Image
Figure Caption
Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and
ZFIN has permission only to display this image to its users.
Additional permissions should be obtained from the applicable author or publisher of the image.
Full text @ Cell Rep.