Fig. 1: Efferocytosis by Rubcn-deficient macrophages induces Notch activation.
From: Exclusion of Notch from the contact site during efferocytosis restricts anticancer immunity
a, Scheme of SLAM-seq. WT or Rubcn−/− BMDMs were cultured with 100 μM s4U in the presence or absence of apoptotic Jurkat cells and incubated at 37 °C for 2 h. Apoptotic cells (ACs) were then washed off with warm complete medium. Cells were cultured for another 2 h in the presence of 100 μM s4U at 37 °C. Total RNA was extracted for library construction and sequencing. b, GSEA of IFN signaling and Notch signaling genes in WT and Rubcn−/− BMDMs upon efferocytosis. GO, Gene Ontology. c, Schematic of experimental design. Apoptotic Jurkat cells expressing tdT were injected i.p. into WT and Rubcn−/− mice. Then, 3 h later, peritoneal macrophages were isolated and subjected to FACS sorting tdT− or tdT+ (gated on DAPI−CD45+CD11b+F4/80+) for RNA-seq. d, GSEA of upregulated Notch1 target genes in WT and Rubcn−/− peritoneal macrophages upon efferocytosis as in c. e, Western blot of nuclear N2ICD in WT, Rubcn−/− and Atg5−/− BMDMs that had engulfed apoptotic HT115 cells or not. TATA-binding protein (TBP) and apoptotic peptidase-activating factor1 (APAF1) served as markers for nuclear and cytosolic fractions, respectively. f, Western blot of nuclear N2ICD in WT and Rubcn−/− BMDMs with or without 1 μM CytoD treatment for 3 h upon efferocytosis. g, Confocal microscopy imaging of WT and Rubcn−/− RAW264.7 cells expressing Notch1–mCherry–NLS reporter and TIM4. Cells were cultured with unconjugated (control) beads or beads conjugated with DLL1 ± biotinylated PS for 3 h before fixing and imaging. Activation of Notch1 receptor was quantified by the intensity of mCherry in the nucleus over the total for each cell (100 × nuclear/total). Control group: WT, n = 21; Rubcn−/−, n = 25. DLL1 group: WT, n = 33; Rubcn−/−, n = 20. DLL + PS group: WT, n = 28; Rubcn−/−, n = 52. Statistical analysis was conducted using a Student’s t-test. Scale bars, 10 μm. h, WT RAW264.7 cells expressing Notch1–mCherry–NLS reporter and TIM4 were cultured with the indicated beads for 3 h before fixing and imaging. For RAW264.7 cells incubated with a mixture of beads coupled with DLL1 plus PS (no color) and DLL1 alone (green), cells that had engulfed or were in close contact with both were quantified as described above. Control, n = 33; DLL1, n = 38; DLL1 + PS, n = 34; DLL1 + PS and DLL1, n = 30; DAPT, n = 28. Asterisks indicate engulfed beads. Data are the mean ± s.d. Dots represent single cells. Statistical analysis was conducted using a Student’s t-test. Scale bars, 10 μm. GSEA significance was calculated using one-sided permutation testing based on a preranked approach. Panels created in BioRender: a, Verbist, K. https://biorender.com/jsrbskx (2026); c, Verbist, K. https://bioRender.com/q7evtdr (2026).
