Figure - PMC

Skip to main content

An official website of the United States government

Here's how you know

Here's how you know

Official websites use .gov
A .gov website belongs to an official government organization in the United States.

Secure .gov websites use HTTPS
A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

View full-text article in PMC

. 2018 Oct 15;7:e40474. doi: 10.7554/eLife.40474

Search in PMC
Search in PubMed
View in NLM Catalog
Add to search

© 2018, Zago et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

PMC Copyright notice

Figure 5. — (A) Comparison of invasive capability of HEK-HT and HEK-HT-H-RasV12 cells through a thin matrigel layer using Transwell invasion assay (6 hr). Representative images of invading cells stained with DAPI are shown below the graph. Scale bar, 50 µm. Graph shows the mean ±SEM. Each dot represents one experiment. For statistics Student t-test was used. (B) Comparison of invasive capability of HEK-HT and HEK-HT-HRasV12 cells through a 3D bovine collagen gel (2.3 mg/ml) using Inverted invasion assay (4 days). Cells were live stained with CellTrace. Invasion was quantified as mean fluorescence of confocal fields acquired every 5 µm starting from below the porous membrane till 200 µm distance inside the gel. Graph shows the mean ±SD of measurements in duplicate from three experiments. Representative images are shown below the graph. Scale bar, 250 µm. Statistical comparison of the curves was done using Student t-test at every 25 µm. (C) RalB but not RalA is required for HEK-HTRasV12 invasion through a matrigel layer (Transwell invasion assay). Cells were transfected with the indicated siRNAs. For statistics one-way ANOVA test was used. (D) RalB is required for HEK-HTRasV12 invasion through a 3D collagen gel (Inverted invasion assay). Invasion was quantified as in panel B. (E) Comparison of invasive capability of HEK-HT-H-RasV12, HEK-HT-H-RasV12G37 and PIK90-treated HEK-HT-H-RasV12G37 cell lines using Transwell invasion assay. The combination of the additional G37 mutation and treatment with the PI3K inhibitor PIK90 (10 µM, treatment started 1 hr before Transwell invasion assay and maintained during the 6 hr invasion) impairs RasV12-dependent MAPK and PI3K hyperactivation, conserving only the activation of Ral pathway. For statistics one-way ANOVA test was used. (F) Comparison of invasive capability of HEK-HT-H-RasV12 cells treated with DMSO, with the MEK inhibitor Trametinib (10 nM), with the PI3K inhibitor PIK90 (2.5 μM), or with both Trametinib and PIK90. Treatments were started 1 hr before Transwell invasion assay and maintained during the 6 hr invasion. Cell lysates were prepared at 1 hr post-treatment. On the right, representative western blots for total Akt and phospho-Akt (Ser437), and for total Erk and phospho-Erk (Thr202/Tyr204), show the efficient pharmacological blockage of MAPK and PI3K pathways. Quantifications of Akt and Erk phosphorylation, calculated as p-Akt/total Akt or p-Erk/total Erk ratios, and normalized for HEK-HT-HRasV12 without drugs, are shown below the WBs. For statistics one-way ANOVA test was used. *p<0.05, **p<0.01, ***p<0.001, ns not-significant.

Figure 5—figure supplement 1. — (A) Comparison of invasive capability of HEK-HT and HEK-HT-H-RasV12 cells through a thin matrigel layer using Transwell invasion assay (6 hr). Representative images of invading cells stained with DAPI are shown below the graph. Scale bar, 50 µm. Graph shows the mean ±SEM. Each dot represents one experiment. For statistics Student t-test was used. (B) Comparison of invasive capability of HEK-HT and HEK-HT-HRasV12 cells through a 3D bovine collagen gel (2.3 mg/ml) using Inverted invasion assay (4 days). Cells were live stained with CellTrace. Invasion was quantified as mean fluorescence of confocal fields acquired every 5 µm starting from below the porous membrane till 200 µm distance inside the gel. Graph shows the mean ±SD of measurements in duplicate from three experiments. Representative images are shown below the graph. Scale bar, 250 µm. Statistical comparison of the curves was done using Student t-test at every 25 µm. (C) RalB but not RalA is required for HEK-HTRasV12 invasion through a matrigel layer (Transwell invasion assay). Cells were transfected with the indicated siRNAs. For statistics one-way ANOVA test was used. (D) RalB is required for HEK-HTRasV12 invasion through a 3D collagen gel (Inverted invasion assay). Invasion was quantified as in panel B. (E) Comparison of invasive capability of HEK-HT-H-RasV12, HEK-HT-H-RasV12G37 and PIK90-treated HEK-HT-H-RasV12G37 cell lines using Transwell invasion assay. The combination of the additional G37 mutation and treatment with the PI3K inhibitor PIK90 (10 µM, treatment started 1 hr before Transwell invasion assay and maintained during the 6 hr invasion) impairs RasV12-dependent MAPK and PI3K hyperactivation, conserving only the activation of Ral pathway. For statistics one-way ANOVA test was used. (F) Comparison of invasive capability of HEK-HT-H-RasV12 cells treated with DMSO, with the MEK inhibitor Trametinib (10 nM), with the PI3K inhibitor PIK90 (2.5 μM), or with both Trametinib and PIK90. Treatments were started 1 hr before Transwell invasion assay and maintained during the 6 hr invasion. Cell lysates were prepared at 1 hr post-treatment. On the right, representative western blots for total Akt and phospho-Akt (Ser437), and for total Erk and phospho-Erk (Thr202/Tyr204), show the efficient pharmacological blockage of MAPK and PI3K pathways. Quantifications of Akt and Erk phosphorylation, calculated as p-Akt/total Akt or p-Erk/total Erk ratios, and normalized for HEK-HT-HRasV12 without drugs, are shown below the WBs. For statistics one-way ANOVA test was used. *p<0.05, **p<0.01, ***p<0.001, ns not-significant.