Figure 4

GTP-binding deficienttransglutaminase 2 fails to promote survival, invasion and drug resistance. (A) A transwell Matrigel invasion assay was performed with the indicated MCF10A sublines. Cells that invaded through the Matrigel after 72 hours of incubation were counted in five random microscopic fields. Original magnification ×20. Experiments were performed three times in triplicate. The results shown are the average number of invading cells per field ± SEM. Vec, lentiviral vector. (B) The sensitivity of the indicated MCF10A sublines to doxorubicin is shown. Quadruplicate wells in 96-well plates, containing 2,000 cells per well in 0.2 ml of the complete medium (10% FCS), were either left untreated or treated with the indicated concentrations of doxorubicin. Two days after the treatment viable cells remaining in the wells were determined by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) reduction test, and percentage cell viability was calculated. Experiments were repeated at least three times with similar results. Bars, mean of quadruplicate values from a representative experiment; lines, SD. (C) Average number of colonies formed from three independent experiments ± SEM after 3 weeks of incubation of the cells in soft agar. Images of colonies formed from a representative experiment after 3 weeks of culture in soft agar are shown in the right panel. Original magnification ×10. (D) Phase-contrast images of acinar structures (at days 4 and 12) formed as a result of the indicated MCF10A cells cultured in Matrigel-coated glass slide chambers for the indicated time periods. (E) Loss of basement membrane in MCF10A sublines expressing WT- or C277S-TG2 after 12 days of culture in Matrigel-coated chambers. Structures thus obtained were immunostained for laminin V (green) and 4',6-diamidino-2-phenylindole (blue). Images shown are from a representative experiment repeated twice with similar results. Original magnification ×20.