Fig. 3

TBX3 overexpression in DCIS-like 21NT cells results in a more aggressive phenotype in 3D Matrigel. TBX3 transfectants were seeded in 3D Matrigel at 3000 cells per well and incubated at 37 °C and 5 % CO₂ for 9 days. a Immunofluorescence images depicting colony size of 21NT transfectants. Cells were grown in Matrigel for 9 days, then stained with Hoechst and Phalloidin and imaged using 60X objective. Scale bars represent 20 μm. b Brightfield and phase contrast images showing growth of colonies after 9 days in 3D Matrigel. Brightfield images were taken using 4X objective, with the scale bar representing 100 μm. Phase contrast images (inset) were taken using 40X objective, with the scale bar representing 20 μm. c TBX3 overexpression increases colony formation rates. A colony was considered to be successfully formed when larger than 50 μm in diameter. Analysis was conducted using ImageJ. d TBX3 overexpression decreased the proportion of round colonies. Analysis was conducted using ImageJ. A binary quantification method was utilized; a circularity index above 0.75 was considered circular. e TBX3 overexpression increases the number of nuclei per cell colony. f TBX3 overexpression resulted in an increase in the percentage of Ki-67 positive cells within colonies. g TBX3 overexpression resulted in a decrease in the percentage of cleaved caspase 3 positive cells within colonies. h The proliferation/apoptosis ratio, defined as the percentage of positive Ki67 cells divided by the percentage of positive cleaved caspase three cells, increased with overexpression of both TBX3 isoforms. Means were analyzed using one-way ANOVA followed by Tukey’s post hoc test. The proportion of circular colonies was analyzed using Fisher’s exact test. A value of p < 0.05 was considered statistically significant; *p < 0.05, **p < 0.01, ***p < 0.001. Error bars indicate Standard Error. Results are representative of at least three independent experiments