Article Figures & Data
Figures
- Figure 1.
Megf10 is necessary for apoptotic cell uptake by astrocytes, and its deficiency results in accumulation of apoptotic cells in the developing CB. A, Megf10 mRNA expression in different organs from C57B6 WT male mice as measured by RT-PCR (n = 3). B, Megf10 expression in the CB of P7 WT, Megf10+/−, and Megf10−/− mice (n = 3–5; p < 0.01). C, Quantification of anti-CC3 staining in the CB of WT, Megf10+/−, and Megf10−/− P7 mice showing that Megf10+/− and Megf10−/− mice had a significant accumulation of apoptotic cells in the CB compared with WT littermates (*p < 0.05, **p < 0.01; WT, n = 4 mice; Megf10+/−, n = 9 mice; Megf10−/−, n = 3 mice). D, Representative images of anti-CC3 staining quantified in C. Scale bars: 100 μm; insets, 1 μm. E, TUNEL staining of the same CB described in C. F, Cultured Megf10+/− and Megf10−/− astrocytes have reduced engulfment of apoptotic MEFs prelabeled with YoPRO-A488 (*p < 0.05, **p < 0.01; n = 9). G, A representative histogram of the experiment quantified in F. H, I, Representative confocal pictures of prelabeled apoptotic CAD neurons engulfment by HEK-293T cells transfected with RFP (H) or mMegf10–RFP (I). J, K, Transfected HEK cells were incubated with prelabeled apoptotic CAD neurons (J) or MEFs (K). Scale bar, 10 μm. Phagocytosis is quantified by counting the percentage of cells containing an engulfed nucleus in three independent experiments done in triplicates (*p < 0.05; ***p < 0.001).
- Figure 2.
Megf10 does not bind directly PS but binds C1q with high affinity. A, Schematic depiction of secreted ex-hMegf10 tagged with His and Flag tag. Western blot confirmed that the secreted and purified protein is a single 100 kDa band as predicted. B, Dot blots showing binding of ex-hMegf10 to various membrane lipids and no binding to PS. C, Quantitative densitometry of the blot presented in B (***p < 0.0001; n = 3 from 3 independent experiments). D, Representative dot blot of binding of ex-hMegf10 to immobilized C1q and quantitative densitometry showing a significant increase in binding compared with secondary antibody alone (*p < 0.05; n = 4). E–G, Label-free surface plasmon resonance confirming that there is no binding to PS (F) and showing affinity of ex-Megf10 to C1q in the nanomolar range with a highly significant (χ2 < 1) as measured by full kinetic analysis (G). H, HEK expressing control GFP or full-length hMegf10–GFP (FL M10-GFP) were incubated with the indicated amounts of Alexa-Fluor 647–C1q (C1q-647) for 1 h and measured by flow cytometry. FL M10 analysis was done on the GFP-positive transfected population (FL M10-GFP+) and the GFP-negative nontransfected population (FL M10-GFP−; n = 3). I, J, HEK cells expressing unlabeled hMegf10 were incubated with C1q-647 (red) (I) or C1q-647 (red) and apoptotic cells (white) (J), washed, and stained with rabbit anti-Megf10 antibody (green) and DAPI nuclear stain (blue). HEK–Megf10 cells take up C1q-647 (I) and apoptotic cells coated with C1q-647 (J). Scale bar, 20 μm.
- Figure 3.
EMARDD mutations impair engulfment of apoptotic cells. A–D, Anti-Megf10 staining of HEK cells expressing control (A), full-length hMegf10 (B), hMegf10–C326R (C), and hMegf10–C774R (D) validating expression of Megf10 on the cell surface. E, Mean fluorescent intensity (MFI) of GFP-positive cells confirm similar expression levels of Megf10 in all transfected cells. F, Analysis of UV MEF uptake by HEK transfected cells shows a defect in engulfment in hMegf10 mutations C326R (22% reduction) and C774R (53% reduction) compared with full-length hMegf10 (***p < 0.0001; n = 9 from 3 independent experiment run in triplicates). G, H, Transfected HEK cells were incubated with the indicated amounts of Alexa-Fluor 647–C1q for 1 h. Percentage of uptake (H) and MFI (I) was measured by flow cytometry.
