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Impact of the 3D Microenvironment on Phenotype, Gene Expression, and EGFR Inhibition of Colorectal Cancer Cell Lines

Figure 6

Influence of culture conditions on EGFR signaling molecules and EGFR inhibition of CRC cells.

A) Schematic representation of the EGFR signalling pathway. B) Immunoblot analysis of AKT, phospho-AKT (S473), p44/42 MAPK and phospho p44/42 MAPK (Thr202/Tyr204). Equal amounts of total protein isolated from cells cultivated as 2D or lrECM 3D cultures were analyzed by SDS/PAGE/immunoblotting as indicated. β−actin served as loading control. SKBR3 served as a positive control for the expression of EGFR signaling molecules. C) Expression of the EGFR protein in 2D or lrECM (3D) cultivated CACO-2, DLD-1, HT-29, SW-480, LOVO, COLO 205 and COLO-206F cells. Cell lysate (40 µg protein per lane) was analyzed by SDS/PAGE/immunoblotting using monoclonal anti EGFR antibody. β-actin served as loading control. D–G) Treatment of 2D or lrECM cultivated CRC cells with AG1478. Two-tailed P-values were calculated by the paired t-test (** indicates a P-value <0.001; *** indicates a P-value <0.0001; ns = not significant). CACO-2 cells were treated for 48 h (D) or 96 h (E) with different concentrations of AG1478 as indicated. F–G) DLD-1 and HT-29 cells were incubated with the EGFR inhibitor AG1478 for 48 h.

Figure 6