Figure 6
GTP-binding function of transglutaminase 2 is essential for promoting the stem cell phenotype. (A) Fluorescence-activated cell sorting analysis for the mammary stem cell antigenic markers CD44 and CD24 in MCF10A cells expressing catalytically inactive (TG2-C277S) and GTP-binding-defective (TG2-R580A) forms of transglutaminase 2 (TG2). (B) Phase-contrast images of mammospheres formed by the indicated MCF10A cells (upper panel). In vitro quantification of mammospheres formed by TG2-C277S- and TG2-R580A-transfected MCF-10A cells (lower panel). The data shown are the number of mammospheres formed per 1,000 seeded cells ± SEM. (C) In vitro quantification of mammospheres formed by TG2-C277S-expressing MCF10A cells at each serial passage (M1 to M4). (D) In vitro quantification of mammospheres formed by TG2-R580A-transfected MCF10A cells after different serial passages (M1 to M4). The data shown are the average number of mammospheres formed per 1,000 seeded cells ± SEM of triplicate values from a representative experiment. Note the difference in scale (600 vs 10) for (C) and (D). (E) Phase-contrast images of differentiated mammospheres following their 12-day culture in Matrigel in the presence of prolactin. (F) The differentiated structures were immunostained for luminal marker Muc1 (red), for basal marker CD49f/integrin α6 (green) and for nuclei with 4',6-diamidino-2-phenylindole (blue).