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. 2015 Mar 4;17(2):155–166. doi: 10.1016/j.neo.2014.12.003

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© 2014 Neoplasia Press, Inc. Published by Elsevier Inc.

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

PMC Copyright notice

NHE1 drives basal and EGF-driven 3D colony growth and invasive behaviors.

(A) Representative images of colonies from the four PDAC cell lines entrapped in 7% Matrigel plus/minus 100 ng/ml EGF and plus/minus 10 μM cariporide. Images were taken at different days after plating (indicated to the right of images) because of the large differences in colony growth rate. Scale bar, 200 μm.

(B) Colony growth rates as increase in colony volume per day calculated in ImageJ. Mean ± SEM, unpaired Student’s t test. *P < .01, **P < .001, and ***P < .0001 (n = 5) compared with the control cells and ^††P < .001 and ^†††P < .0001 compared to the respective EGF cells for each line.

(C) Colony circularity index measured and calculated in ImageJ. Mean ± SEM, unpaired Student’s t test. *P < .01, **P < .001, and ***P < .0001 compared with the control cells and ^††P < .001 and ^†††P < .0001 compared to the respective EGF cells for each line.

(D) Invasion across a Matrigel layer in Boyden chambers. Error bars, ± SEM. *P < .01, **P < .001, and ***P < .0001 compared with the control cells and ^†P < .01, ^††P < .001, and ^†††P < .0001 compared to the respective EGF cells (n = 3).

(E) Invadopodia focal ECM proteolysis in PANC-1 cells. Representative images show focal ECM proteolysis (green) and actin immunofluorescence (red). The insets show enlarged views of the boxed regions in which F-actin dots colocalize with focal fluorescence (arrows) released after the cells had digested the ECM. Scale bars, 20 μm for micrographs, 10 μm for inset. See also Figure S3.

(F) Quantification of the invadopodia focal digestion activity in response to cariporide, EGF, or their combination; error bars, ± SEM. *P < .01 and **P < .01 compared with the control cells and ^†P < .01, ^††P < .001, and ^†††P < .0001 compared to the respective EGF cells (n = 5).

(G) Motility expressed as total distance traveled (translocation). Error bars, ± SEM. *P < .05, **P < .01, and ***P < .001 compared with the control cells and ^†P < .05, ^††P < .001, and ^†††P < .0001 compared to the respective EGF cells (n = 8-11 experiments). See also Figure S4.