Extended Data Figure 5. The role of SLC2A1 for efferocytosis.
(a) Slc2a1fl/fl BMDM were treated with or without Tat-Cre to delete Slc2a1. The cells were then incubated with IgG-coated Jurkat cells and engulfment was assessed by CypHer5E signal within the BMDM. The uptake by the control BMDM (not treated with Tat-Cre, and denoted WT) were set to 1.
(b) siRNAs targeting of Slc2a1 down-modulates SLC2A1 protein expression. Shown are representative western blots of siRNA knockdown of Slc2a1 in LR73 cells versus scrambled siRNA. Also shown are LR73 cells expressing siRNA-resistant SLC2A1.
(c) Slc2a1 deletion efficiency in Cas9-LR73 cells. Slc2a1 guide was introduced into Cas9-EGFP+ LR73 cell clones. The efficiency of Slc2a1 deletion was quantified using qPCR.
(d) Introduction of TAT-Cre into Slc2a1flfl bone marrow-derived macrophages efficiently knocks down SLC2A1 protein expression. Slc2a1fl/fl bone marrow cells were treated with recombinant TAT-Cre during macrophage differentiation after isolation from the bone marrow.
(e) STF-31 did not affect antibody-mediated phagocytosis by peritoneal macrophages. C57BL/6 mice were intraperitoneally injected with 10mg/kg of either STF-31 in X-VIVO media 1h prior to IgG-coated Jurkat cell injection. CypHer5E labeled Jurkat cells were injected intraperitoneally along with the drug. Mice were euthanized 1h later, peritoneal cells collected, and apoptotic cell engulfment by CD11b+ F4/80hi macrophages was analyzed by FACS.
(f) Slc2a1-deficient LR73 cells or BMDMs were treated with STF-31, and the engulfment assay was conducted using CypHer5E-labeled apoptotic Jurkat cells. The CypHer5E+ phagocytic cells after 2h of incubation was determined by flow cytometry. n.s., not significant. Data are representative of at least two independent experiments with 3–4 replicates per condition.
(g) The SLC2A1 inhibitor STF-31 does not increase 7AAD+ thymocytes in vitro. Isolated thymocytes were incubated with dexamethasone (10μM) with or without STF-31 (2mM). 4h later, the thymocytes cell death were addressed by Annexin+ 7-AAD+. Data are representative of two independent experiments.