FIG. 7.

RANKL directly induces proliferation and lumen repopulation in MMTV-RANK acini. (A) Phase-contrast microscopy of WT and MMTV-RANK primary MEC 3D cultures, without or with RANKL (RL) at day 4. Scale bar, 100 μm. The images are representative of five experiments. Note the significant change in morphology of the MMTV-RANK acini in the presence of RANKL. (B) Size of WT and MMTV-RANK acini after 5 days in culture in the absence (−) or presence (+) of RANKL (RL). The upper and lower bars indicate the 90th and 10th percentiles of the data. The data points greater than the 90th or less than the 10th percentiles are plotted as individual dots. For each treatment 12 to 92 spheroids were measured. (C) Confocal images at day 5 of WT and MMTV-RANK mammary acini cultured without or with RANKL (+RL) as indicated. Nuclei were stained with DRAQ5 (blue) and Ki-67 or activated caspase 3 (green). Arrows indicate Ki-67-positive cells. One or more Ki-67-positive cells was detected in 30 to 50% of the WT acini and in 100% of the MMTV-RANK acini cultured with RANKL. Representative images are shown. Scale bar, 50 μm. Note the mislocalized or sparse apoptotic cells detected in MMTV-RANK acini cultured with RANKL and the presence of a filled lumen.