% of max Supplementary Figure 1 Control GST GST-RP 17 kda α2-mg 42 kda b-actin Gate: CD11c+ (DCs) Gate: F4/8+ (Mfs) IgG Cd11cCre + Lrp1 fl/fl LRP-1 Supplementary figure 1. () MDCs were pretreated with μm chloroquine to prevent degradation of internalized proteins and then incubated with methylamine-activated α2-macroglobulin (α2-mg) for 3 min in the presence of vehicle control (PS), GST (1 μg/ml), or GST-RP (1 μg/ml). The internalized α2-mg was detected by immunoblot using an antibody against α2-mg (top blot). β-actin was used as a loading control (bottom blot). () nalysis of expression levels of LRP-1 in splenic DCs (left panel, CD11c+ gate) or splenic macrophages (right panel, F4/8+ gate) in (blue line) and Cd11cCre + Lrp1 fl/fl mice (DC-LRP1 KO; red line), respectively. The green line represents cells stained with an IgG control antibody.
% C uptake by splenic Mφs % C uptake by splenic Mφs Supplementary Figure 2 9 8 7 6 4 3 2 1 CD36 9 8 7 6 4 3 2 1 9 8 7 6 4 3 2 1 9 8 7 6 4 3 2 1 1 8 6 4 2 xl KO 8 7 6 4 3 2 1 9 8 7 6 4 3 2 1 9 8 7 6 4 3 2 1 Supplementary figure 2. In-vivo efferocytosis by splenic macrophages. Similar to Figure 2, except the FCS analysis was gated on F4/8 + cells to quantify uptake of PKH67- labeled Cs by splenic macrophages. LRP-1 was inhibited by i.v. injection of GST-RP (2 mg/mouse), and Tim-3 was inhibited by i.p. injection of a neutralizing anti-tim-3 antibody (RMT3-23, 1 μg/mouse). n=3 mice per group., p<.;, no significant difference.
Mean fluorescence intensity % efferocytosis 3 3 Supplementary Figure 3 2 2 1 1 # # # xl -/- xl -/- xl -/- xl -/- IgG + RP + Tim3 nb + + + Scarf1 nb + + + + IgG 6 4 3 Scarf1 nb 2 1 IgG Scarf1 nb 1 2 Supplementary Figure 3. () MDCs from or xl -/- mice were treated with. µg/ml IgG or neutralizing antibody (nb) against Tim3 or Scarf1 (Proteintech) for 3 min prior to addition of PKH67- labeled Cs. Efferocytosis was measured 2 h later by microscopic analysis., p<.; #, p,. as compared with its appropriate control;, no significant difference. () Related to Fig. 3, positive control for Scarf1 nb in human splenic DCs. Human splenic DCs were treated with. µg/ml IgG or Scarf1 nb (. μg/ml) (R&D systems) for 3 min prior to addition of DiI-labeled acetylated low-density lipoprotein (DiI-c-LDL, 1 μg/ml). The uptake of DiI-c-LDL was measured by microscopic analysis (red staining); the right panels show the phase images. The quantified data of c-ldl uptake measured by analysis of mean fluorescence intensity is shown in the bar graph. n=3., p<..
Count % efferocytosis Supplementary Figure 4 MDCs 2 2 1 1 CD11c+ gate Cd11cCre + Lrp1 fl/fl CD11c+ gate xl -/- xl LRP-1 Supplementary Figure 4. () MDCs from Lrp1 fl/fl or Cd11cCre + Lrp1 fl/fl mice were incubated with PKH67-labeled Cs for 2 h at 37 C with or without neutralizing antibodies against CD36 and αv integrin. Efferocytosis was quantified as the percentage of MDCs with internalized Cs., p<.;, no significant difference. () The histogram plots are representative data of expression levels of xl (left panel) and LRP-1 (right panel) in splenic DCs (CD11c + gate) of the indicated groups of mice. n=3 mice per group.
% of max Count Count Supplementary Figure IP: LRP-1 LRP1-46 (donor) xl-647 (acceptor) xl-46 (donor) LRP-647 (acceptor) 84 kda LRP-1 Control Control Lrp fl/fl 14 kda xl xl -/- CD11cCre + Lrp fl/fl FRET channel (sensitized emission) C Control LC-46 LC-46+ HC-647 Control LC-46 LC-46+ HC-647 Senstized emission Donor fluorescence quenching Supplementary figure. () Immunoprecipitation of LRP-1 from whole cell lysates of MDCs obtained from or xl -/- mice followed by Western blotting to detect LRP-1 (top panel) or xl (bottom panel). () Flow cytometric analysis of FRET (sensitized emission) between LRP-1 and xl, using either LRP-1 as donor (LRP-1-46) in splenic DCs (CD11c + gate) of and xl -/- mice (left panel), or using xl as donor (xl-46) in and Cd11cCre + Lrp1f l/fl mice (right panel). IgG conjugated to lexa fluor 46 was used as control (orange line). (C) FCS-FRET analysis between MHC-I heavy and light chain was used as a positive control. Shown are sensitized emission (left panel) and donor fluorescence quenching (right panel) in DCs stained with an antibody against MHC-I light chain conjugated to a donor fluorophore and antibody against MHC-I heavy chain conjugated to an acceptor fluorophore.
CD11c hi cells (% total splenocytes) CD8α+ cells (% CD11c hi splenocytes) % C uptake % of max % of max Supplementary Figure 6 IgG Ranbp9 -/- Ranbp9 -/- Ranbp9 -/- RanP9 xl LRP-1 C 3 2. 2 1. 1. D 3 3 2 2 1 1 RanP9 KO E 9 8 7 6 4 3 2 1 Mφ Supplementary figure 6. () Flow cytometric analysis of RanP9 expression in splenic DCs (CD11c + gate) of chimeric mice generated by transplantation of (blue line) or Ranbp9 -/- (green line) fetal liver cells. The data are representative of 3 independent experiments. () nalysis of cell surface expression of xl and LRP-1 in CD11c hi -gated splenocytes derived from mice transplanted with fetal liver from (blue line) or Ranbp9 -/- (red line) embryos. (C-D) The bar graphs show the percent CD11c hi cells of total splenocytes and the percent CD8α + of total CD11c hi cells in and Ranbp9 -/- mice. (n=3; n.s, no significant difference). (E) The bar graph shows quantified data of splenic macrophage efferocytosis in or Ranbp9 -/- chimeric mice 3 h post-injection of PKH67-labeled Cs. n=4 mice per group., no significant difference.
% efferocytosis Supplementary Figure 7 3 MDC 2 2 1 1 Con sigulp xl -/- Con xl -/- sigulp Supplementary figure 7. MDCs from or xl -/- mice were transfected with negative control scrambled RN (Con) or sirn against GULP1 (sigulp1). Efferocytosis was assayed 48 h later by incubating the cells with PKH67-labeled Cs for 2 h at 37 C. Efferocytosis was quantified as the percentage of MDCs with internalized Cs., p<.;, no significant difference.
+ HSV1 xl -/- + HSV1 Lrp1 fl/fl + HSV1 Cd11cCre + Lrp1 fl/fl + HSV1 Ranbp9 -/- + HSV1 drenal weight (mg) Supplementary Figure 8 Gate: CD8+CFSE+ xl -/- 14 12 1 8 6 4 Lrp1 fl Cd11cCre + Lrp1 2 fl Ranbp9 -/- 1 2 3 4 6 7 8 9 1 xl -/- Lrp1 fl/fl Cd11cCre + Lrp1 fl/fl Ranbp9 -/- Supplementary figure 8. () CFSE dye-dilution analysis of CD8 + -gated gt-i.1 transgenic T cells in the indicated groups of mice 4 days post-infection with HSV-1. n=3 mice per group. () Weights of adrenal glands of the indicated groups of mice 4 days post-infection with HSV-1. n=3 mice per group., p<.;, no significant difference.
Pfu (x1 )/ml cell extract Pfu (x1 )/ml cell extract Supplementary Figure 9 MDC Primary adrenal cells 3. 3 2. 2 1. 1. 7 6 4 3 2 1 1 2 3 1 2 Supplementary figure 9. MDCs (left panel) or primary adrenal cells (right panel) from, xl -/-, or Cd11cCre + Lrp1 fl/fl mice were infected with HSV-1 at an MOI of 2 pfu/cell. On day 2 postinfection, the cells were lysed, and viral titers were determined by quantifying plaque forming units in Vero cells., p<.;, no significant difference (n=3).
% C uptake by splenic DCs in vivo % un-divided gt-i.1 T cells Supplementary Figure 1 3 2 2 1 1 7 6 4 3 2 1 1 2 3 DC-LRP1 KO Supplementary figure 1. () xl -/- or Cd11cCre + Lrp1 fl/fl mice were injected with PKH67-labeled apoptotic HSV-1 infected primary adrenal cells (3x1 6 cells/mouse) and then splenic DCs were assayed for efferocytosis. () Lrp1 fl/fl or Cd11cCre + Lrp1 fl/fl mice were injected with CFSElabeled CD8 + gt-i.1 Tg T cells (1 6 cells/mouse) and UV-inactivated HSV-1 killed primary adrenal cells (1 6 cells/mouse). On day 4 post-injection, the splenocytes were analyzed by flow cytometry for CFSE dye dilution on the CD8 + CFSE + -gated population., p<. (n=3 mice per group).
HSV-1 pfu (x1 3 )/adrenal IFN-α (pg/ml) Supplementary Figure 11 9 8 7 6 4 3 2 1 xl KO xl -/- Day 7 Day 1 1 8 6 4 2 1 2 3 Supplementary Figure 11. () Viral titer in adrenal glands of and xl -/- mice on day 7 and 1 post-infection with HSV-1 (4x1 pfu/mouse). n=3 mice per group. () ELIS measurement of IFN-α levels in serum of or xl -/- mice on day 4 post-infection with HSV-1 (4x1 pfu/mouse). n=3 mice per group., p<.;, no significant difference.