Figure 6
MerTK directs efferocytosis in mammary epithelial cells. A. Taqman™ real-time PCR was used to quantitate the relative levels of mertk mRNA in mouse mammary total RNA, harvested at the indicated time points. The relative levels of mertk were calculated using expression of mouse GAPDH to correct for total RNA concentration using the δδCT method, setting the value of mertk expression measured at 6 weeks to 1. Average values are shown ± S.D. N = 3, each sample analyzed 6 times. Statistical significance using Student's T-test (unpaired). B. Immunohistochemistry to detect MerTK in WT mammary glands harvested from virgin female mice at 6 and 12 weeks of age. Representative images are shown. C. WT and mertk-/- PMECs were incubated for 4 hours in the presence of live or apoptotic GFP+ cells harvested from the thymus of untreated or dexamethasone-treated (respectively) actin-GFP mice. Representative photomicrographs of WT PMECs ingesting GFP+ apoptotic thymocytes are shown. D. Flow cytometric analysis of WT and mertk-/- PMECs incubated for 4 hours in the presence of apoptotic cells (ACs) or live cells (LCs). N> 30,000 cells examined per genotype. Value in right panel indicates percentage of total cells that were GFP+. E. Real-time RT-PCR was used to measure mertk mRNA expression in whole mammary RNA harvested from 6 weeks virgin female mice, or from female mice at 1, 3, 10, and 60 dpfw. Values shown represent the average relative values, ± S.D. N = 3, each analyzed six times. F. Mammary glands harvested at 16.5 dpc and 1 dpfw were immunostained for MerTK. Representative images are shown. G. Real-time RT-PCR was used to measure mertk mRNA expression in total RNA harvested from WT PMECs cultured alone or for 4 hours in the presence of apoptotic thymocytes for 4 hours. Note that thymocytes were removed with three PBS washes prior to harvesting RNA from PMECs. Where indicated, PMECs were pre-incubated with an inhibitory MerTK-antibody or a control IgG (50 μg/μl). Values shown represent the average relative values, ± S.D. N = 3, each analyzed six times. Statistical significance using Student's T-test (unpaired).