Figure 1.
Microenvironmental stiffness regulates glioma cell proliferation.
Effect of ECM rigidity on proliferation of U373-MG (A) and U87-MG (B) cells. Results represent quantification of n>10,000 cells for at least three substrates per condition by flow cytometry, where the percentage of dividing cells was determined as the average percentage of cells staining positive for BrdU incorporation. *, P<0.05 with respect to 119 kPa.
Figure 2.
ECM rigidity regulates glioma cell cycle distribution.
Effect of ECM rigidity on cell cycle distribution of U373-MG (A) and U87-MG (B) cells. Results represent quantification of n>10,000 cells for at least three substrates per condition by flow cytometry, where the percentage of cells in each phase of the cell cycle was determined as the average percentage of cells staining positive for propidium iodide incorporation. *, P<0.05 with respect to 119 kPa.
Figure 3.
Microenvironmental stiffness regulates expression and phosphorylation of EGFR pathway components.
The expression of activated EGFR, activated Akt and PI3K in U373-MG cells rises with increasing substrate stiffness (A). Similarly, the expression levels of EGFR and Akt in U373-MG cells rise with increasing substrate stiffness (B). Results represent quantification of at least three biological replicates on three separate Western blots, where the relative protein expression levels have been first normalized to the expression of GAPDH and then normalized to the expression level on the stiffest substrate of 119 kPa. Representative blots for each protein are on the right. *, P<0.05 with respect to 119 kPa.
Figure 4.
Stifffness-dependent glioma cell proliferation is dampened upon treatment with 20 uM EGFR inhibitor - Tyrphostin, 20 uM Akt inhibitor - Triciribine, and 20 uM PI3 Kinase inhibitor - Wortmannin for 24 hours as compared with the DMSO negative control.
Results represent quantification of n>10,000 cells for at least three substrates per condition by flow cytometry, where the percentage of dividing cells was determined as the average percentage of cells staining positive for BrdU incorporation *, P<0.05 with respect to 119 kPa for DMSO control, 20 uM Tyrphostin and 20 uM Triciribine.
Figure 5.
Colocalization of focal adhesion of phospho-EGFR.
U373-MG cells were cultured on soft (A, C, E) or stiff (B, D, F) polyacrylamide hydrogels and immunofluorescently stained for vinculin (A, B) and phospo-EGFR (C, D). There are no punctate vinculin-positive focal adhesions on soft substrates (A), while there are large focal adhesions on stiff substrates (C). On stiff substrates, there are distinct, punctate pEGFR structures (D; arrows) that colocalize with vinculin positive adhesions (F; arrows). The colocalization is more clearly evident in the high-magnification insets (B, D). Scale bar is 50 microns.