Extended Data Fig. 10: HFSC-mediated apoptotic corpse clearance is necessary to preserve tissue homeostasis.

a, Multiplexed iterative immunofluorescence for T-cell markers on Rxra wild type (WT) and conditional knockout (cKO) skin. Itga6, integrin-α6. Scale bar, 50 um. Images representative of 15 HFs/mouse, 2 mice/genotype. b, Multiplexed iterative immunofluorescence for CD11c⁺ dendritic cells (arrowheads) and F4/80⁺ macrophages on Rxra WT and cKO skin. Images representative of 15 HFs/mouse, 2 mice/genotype. Additional characterization/quantification of macrophage phenotype in Fig. 5a. Scale bar, 30 um. c, Top, Sagittal immunofluorescence of Langerhans cells in the upper HF (above the sebaceous gland) in Rxra WT and cKO skin. Scale bar, 10 um. Bottom, Quantifications of Langerhans cells (LCs) in the dermis (left), MHCII-high Langerhans cells in the skin (middle), and Langerhans cell volume (right) in Rxra WT and cKO skin (n = 213 [WT] and 146 [cKO] Langerhans cells). n = 3 mice per genotype. d, Left, Sagittal immunofluorescence for phospho-STAT3 (pSTAT3) and quantifications (right) in Rxra WT and cKO HFs. Scale bar, 10 um. n = 27 HFs across 3 mice per genotype. e, Left, Sagittal immunofluorescence and quantifications (right) for cJun⁺ HFSCs in Mertk WT and KO HFs. Scale bar, 15 um. n = 39 HFs (WT) and 40 HFs (KO) across 3 mice per genotype. f, Top, Schematic to block apoptotic corpse engulfment by intradermal injection of small molecule inhibitors (Inhib) during catagen followed by quantifications of cJun⁺ HFSCs (bottom) in annexinV (AnxV)-treated skin (to mask exposed phosphatidylserine, left) or HX531 treated skin (to inhibit RXRα, right). AnnexinV experiment: n = 37 HFs (VEH) and n = 38 HFs (AnxV) across 3 mice. HX531 experiment: n = 34 HFs per treatment across 3 mice. g, Top left, Heatmap of differentially accessible peaks in late catagen HFSCs between Rxra WT and cKO pooled replicates, annotated as in Extended Data Fig. 9d. Top middle and right, Motif enrichment analysis of the peaks gaining (middle) or decreasing (right) accessibility in Rxra cKO. Bottom left, Bar chart of gene ontology (GO) terms of genes associated to peaks gained in cKO, ordered on false discorvery rate (FDR). Bottom right, ATAC-seq replicate-pooled peak tracks for cell cycle gene promoters in FACS-isolated Rxra WT and cKO CatVII HFSCs, scaled, normalized, and annotated as in Fig. 2f. Peaks with differential accessibility highlighted in light blue. h,i, Left, Experimental design to block corpse engulfment by masking exposed phosphatidylserine (PS) on apoptotic cells with annexinV (AnxV) (h) or by inhibiting RXRα with HX531 (i) in catagen HFSCs versus respective contralateral vehicle (VEH) controls in vivo, followed by HFSC isolation and colony forming assays in vitro. Quantifications of total colony number (middle) and average colony size (right) following 7 days of culture. n = 3 mice per condition averaged across technical triplicates. j, Left, Schematic to conditionally ablate Rxra immediately prior to catagen, and examine the consequences in the subsequent hair cycle. Middle, Representative image of whole back skin of Rxra WT and cKO littermates, with shaved area denoted by dashed line. Right, Saggital immunofluorescence of age-matched skin from Rxra WT and cKO littermates for hair cycle staging. n = 6 WT and n = 7 cKO mice. Data quantified in Fig. 5e. k, Experimental design (left) and quantification (right) of pSTAT3⁺ HFSCs exposed to corpses they can (VEH) or can’t (AnxV, annexinV) engulf or secondarily necrotic debris. n = 3 (Media) or n = 4 each (other conditions) experiments/condition (averaged duplicates). l, Model for how stem cell-mediated apoptotic cell clearance protects the niche for tissue homeostasis. 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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