Extended Data Fig. 2: Notch receptor is proteolytically activated in Rubicon-deficient macrophages during efferocytosis.

(a) Western blot of JAG1 in live and apoptotic Jurkat cells (5 μM ABT-737 and 5 μM S63845 treatment for 45 mins). (b) Immunoblot of nuclear N1ICD in WT, Rubcn^−/− BMDM that had engulfed apoptotic Jurkat cells. Histone3 H3 (H3) and protein disulfide isomerase (PDI) were used as nuclei and ER markers, respectively. (c) Immunoblot of Notch2 in Jurkat cells stably expressing sgRNAs targeting Notch2. (d) Western blot of nuclear N2ICD in WT or Rubcn^−/− BMDM that had engulfed apoptotic Notch2-deficient Jurkat cells as in (c). (e) Quantification of efferocytosis efficiency in WT or Rubcn^−/− BMDM cultured with apoptotic Jurkat cells expressing tdTomato at 37 °C for 3 hours. Apoptotic Jurkat cells were washed off, and the BMDM were collected and stained with DAPI. The efferocytosis efficiency was determined as the frequency of DAPI⁻, tdTomato⁺ cells by flow cytometric analysis. WT (n = 3) and Rubcn^−/−(n = 3). Student’s t test. (f) Western blot of nuclear N2ICD in WT or Rubcn^−/− BMDM with or without 1 μM CytoD treatment during 3-hours of efferocytosis of apoptotic Jurkat cells. For DAPT treatment, cells were pretreated with 1 μM DAPT overnight before the experiment. RACS1 was used as a Golgi marker. (g) GSEA analysis of RNAseq using WT BMDM cultured with apoptotic Jurkat cells in the presence of DMSO or 1 μM CytoD for 3 hours. (h) Immunoblot of N2ICD in WT and Rubcn^−/− BMDM that had been co-cultured with PS-coated beads in the presence or absence of 1 μM CytoD. (i) Immunoblot of N2ICD in WT and Rubcn^−/− BMDM that had been co-cultured with apoptotic Jurkat cells in the presence or absence of 20 μM MG132 for 3 hours. AC, apoptotic cells. GSEA significance was calculated using one-sided permutation testing based on a pre-ranked approach.

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