A role for galectin-3 in promoting lung colonization in ST6GalNAc2-silenced cells. A role for galectin-3 in promoting lung colonization in ST6GalNAc2-silenced cells. A, siNTC- and siST6-transfected 4T1-Luc cells were labeled with CellTracker red or green dyes, respectively, mixed at a 1:1 ratio, and 0.5 × 106 cells were inoculated into the tail veins of BALB/c mice. At 1 and 24 hours after inoculation, the lungs were extracted and imaged by confocal microscopy, and lung retention was quantified. Data shown are mean tumor cell coverage per field of view (fov) from 4 mice in each group ±SEM. Student t test was used to generate P values. Equivalent results were obtained in three independent experiments, including dye swap experiments. Right, representative confocal images. Scale bar, 200 μm. B, shNTC-A and shST6-A 4T1-Luc cells were labeled with CellTracker green or red dyes, respectively, and lung retention quantified as described in A. C, shNTC-A and shST6-A 4T1-Luc cells were transiently transfected with human ST6GALNAC2 (hST6) or vector alone (VEC), labeled with CellTracker dyes, and lung retention quantified as described in A. D, 4T1-Luc cells were transfected with siNTC or siST6 in the presence or absence of Lgals3siRNA (siGAL3) oligonucleotides. Transfected cells were labeled with CellTracker dyes and lung retention quantified as described in A. E, siNTC and siST6-transfected MDA-MB-453 cells were stained for ST6GalNAc2 (green) and the Golgi protein b-COP (red). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bar, 20 μm. F, G, and H, 0.5 × 106 siNTC-, siST6-, or siGAL3-transfected MDA-MB-453 or MDA-MB-231 cells labeled with CellTracker dyes were inoculated into the tail veins of CD1 mice nu/nu mice and lung retention assessed at 1 and 5 hours as described in A. I, 0.5 × 106 231-VEC or 231-ST6 cells labeled with CellTracker dyes were treated for 30 minutes with vehicle (VEH) or 250 μg/mL GCS-100 and inoculated into the tail veins of CD1 mice nu/nu mice that had been pretreated 1 day previously with 250 μg/mL GCS-100. Lung retention was assessed at 1 and 5 hours as described in A. A–I, qPCR analysis of ST6GALNAC2 and LGALS3 mRNA levels and representative confocal images are shown in Supplementary Fig. S4 (4T1-Luc cells) and Supplementary Fig. S5 (human cells). ns, not significant; AU, arbitrary unit. Nirupa Murugaesu et al. Cancer Discovery 2014;4:304-317 ©2014 by American Association for Cancer Research