Extended Data Fig. 7: Corpses secrete lysophosphatidylcholine and free fatty acids to activate RXRα and fully upregulate the phagocytic program in vitro.

a, Quantifications of RXRα⁺ (Top) and RARγ⁺ (Middle) HFSCs 30 min following addition of corpses with (+VEH) or without exposed phosphatidylserine (PS) (+AnxV), or in the presence of TAM-family inhibitor BMS-777607 ( + BMS). Bottom, Percentage of TAM-family⁺;Lysosome^high HFSCs 4 hrs after corpse exposure. RXRα⁺ HFSCs: n = 7 (Media), n = 6 (Corpses+VEH), n = 5 (Corpses+BMS), n = 4 (Corpses+AnxV); RARγ⁺HFSCs: n = 4 (Media), n = 9 (Corpses+VEH), n = 2 (Corpses+BMS), n = 2 (Corpses+AnxV); TAM⁺Lyso^Hi HFSCs: n = 6 (Media, Corpses+AnxV), n = 9 (Corpses+VEH), n = 11 (Corpses+BMS) experimental replicates. b, Immunofluorescence of HFSCs stained for RXRα or RARγ 30 mins after low-titre corpse addition. Insets are RXRα or RARγ alone. Scale bar, 10 um. Representative of triplicate experiments performed n = 3 times. Both addition of retinoic acid (positive controls) and corpses cause increased nuclear fluorescence of RXRα or RARγ (quantified as RXRα⁺ or RARγ⁺, respectively). c, Left, Heatmap of bulk RNA-sequencing of wild type HFSC replicates exposed to corpses with (+Veh, vehicle) or without (+BEL, bromoenol lactone) secreted lysophosphatidylcholine (LPC) and fatty acids (FAs). Colour bar, Z-score normalized expression. Right, Transcript per million (TPM) expression values of selected phagocytic program genes. n = 2 (Media), n = 4 each (Corpses+Veh, Corpses+BEL). Full list in Supplementary Table 5. d, Strategy (top left) and percentages of RXRα⁺ HFSCs (bottom) to manipulate corpse-derived secreted nucleotides (left) (n = 64 VEH HFs, n = 35 Apyrase HFs, 4 mice), sphingosine-1-phosphate (S1P) (middle) (n = 32 VEH HFs, n = 38 MPA08 HFs, 4 mice), or exposed phosphatidylserine, PS, (right) (n = 29 VEH HFs, n = 31 AnnexinV HFs, 4 mice) by intradermal injections. Contralateral back skin was injected with vehicle (Veh) control. Top right, Uninjected versus vehicle-injected percentage of RXRα⁺ cells. n = 62 HFs uninjected, n = 80HFs injected (CatVI) and n = 49 HFs uninjected, n = 83 HFs injected (CatVII) across 8 mice. e, Percentage of RXRα⁺ (left) and RARγ⁺ (right) HFSCs per experiment, 30 min after addition of the indicated recombinant molecules. dATP & dUTP, dNTP; arachidonic acid, AA; 9cRA, 9-cis retinoic acid; ATRA, all-trans retinoic acid. RXRα⁺ HFSCs: n = 14 (Media), n = 6 (dNTPs, LPC), n = 7 each (S1P, AA), n = 3 each (9cRA, ATRA) experimental replicates. RARγ⁺ HFSCs: n = 5 (Media, ATRA), n = 4 each (AA, LPC), and n = 3 (AA + LPC) experimental replicates. f, Percentage of HFSCs responding to recombinant molecules by increasing RXRα⁺ cells (top) or RARγ⁺ cells (bottom). n = 2 replicates per condition, averaged across technical triplicates. One of two independent experiments shown for each quantification. g, Percentage of TAM-family⁺ HFSCs in response to recombinant molecule combinations. One of two independent experiments shown. n = 4 (Media, 9cRA, LPC + AA), n = 6 (LPC, AA), n = 3 (remaining conditions) experimental replicates. h, Quantitative RT-PCR for phagocytic gene transcripts, relative to B2-microglobulin (B2m) levels, and normalized to media-only conditions. Concentrations of indicated added molecules are as in (g). n = 3 experimental replicates, performed in technical triplicates. Data presented as mean ± s.e.m (error bars). Quantifications, pairwise independent Student’s t-tests (2-sided), p-values indicated. n.s. not significant (p > 0.05). Data as box-and-whisker plots; box: first-to-third quartiles and median, whiskers 1.5× inter-quartile range. More details on statistics and reproducibility can be found in the Methods section.

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