Fig. 1: Crbn negatively regulates phagocytosis of apoptotic cells.

a LR73 cells transfected with Crbn were incubated with TAMRA-stained apoptotic cells for 2 h and analyzed by flow cytometry. n = 3 experiments, mean ± SEM (two-tailed unpaired Student t test). b MEFs derived from WT or Crbn^−/− mice or Crbn^−/− MEFs transfected with Crbn were incubated with TAMRA-stained apoptotic cells for 2 h and analyzed by flow cytometry. n = 3 experiments, mean ± SEM. NS, not significant (one-way ANOVA). c, d BMDMs (c, n = 5 mice) or peritoneal macrophages (d, n = 6 mice) derived from Crbn^−/− or WT mice were incubated with TAMRA-stained apoptotic cells for the indicated durations and analyzed by flow cytometry, mean ± SEM. The numbers in the graphs indicate p values (two-way ANOVA). e TAMRA-stained apoptotic cells were peritoneally injected into WT (n = 19 mice) or Crbn^−/− (n = 21 mice) mice. At 15 min after injection, peritoneal exudates were stained with F4/80 and analyzed by flow cytometry. n = 3 experiments, mean ± SEM (two-tailed unpaired Student t test). f Mice were intraperitoneally injected with 250 µg dexamethasone dissolved in PBS. At 4 h after injection, the sizes of the thymi were observed (top) and the number of thymocytes in the thymi was counted (bottom). n = 5 experiments (each dot represents one mouse), mean ± SEM (two-way ANOVA). g The thymi of the mice f were stained with TUNEL. Microscopy images were acquired (left) and the intensity of TUNEL was measured with ImageJ (right). Scale bar, 250 or 50 µm (inset). n = 3 experiments (each dot represents one slide), mean ± SEM (two-tailed unpaired Student t test). h Thymocytes from the indicated mice were incubated with 50 µM dexamethasone for the indicated duration, stained with Annexin V and 7-AAD, and analyzed by flow cytometry. n = 3 experiments, mean ± SEM. NS, not significant (two-tailed unpaired Student t test). i BMDMs from the indicated mice migrating to 100 nM ATP or ATPγS were measured using the transwell cell migration assay. Scale bar, 200 µm. n = 4 experiments, mean ± SEM. NS, not significant (Two-tailed unpaired Student t test).