Single cell RNA sequencing analyses in epdermis-specofof CerS4 knockout and characterization of sebaceous gland-specifc CerS4 knockout mice. (A) Cell counts of cell clusters determined by scRNAseq in two control (Ctr) and two CerS4^epi−/− mice in a replicate resolved resolution. (B) UMAP projection of CerS4-expressing cells in scRNAseq data of control mice. (C) In situ hybridization of CerS4 mRNA expression in back skin sections of control mice. Scale bars 20 µm. (D) RT-qPCR analysis of control FACS-sorted HFSCs, HF progenitors, and IFE progenitor cells and total epidermis (n = 3 mice). (E) Macroscopic images of Ctr and CerS4^SCD3+/− mice. (F) H/E-stained back skin sections of Ctr and CerS4^SG−/− back skin at P47, P58, and 6 mo. Scale bars 100 µm. (G) Quantification of the thickness of the IFE at P58 (n = 10 control, 6 CerS4^SG−/− mice) and 6 mo (n = 5 mice/genotype) from back skin sections. (H) Quantification of the size of SG at P58 (n = 10 mice/genotype) and 6 mo (n = 10 control, 9 CerS4^SG−/− mice) from back skin sections. (I) FACS analysis of CD34⁺ integrin α6⁺ HFSCs from P47 (n = 6 control, 7 CerS4^epi−/− mice), P58 (n = 11 control, 6 CerS4^epi−/− mice), and 6-mo-old mice (n = 11 control, 6 CerS4^epi−/− mice). (J) Gene expression levels of selected stem and progenitor cell marker genes from scRNAseq data in the OB stem cell compartment in Ctr and CerS4^epi−/− skin in a replicate resolved presentation. Note that Ctr and CerS4^epi−/− replicates show comparable changes. (K) GSEA of differentially expressed genes in Ctr and CerS4^epi−/− OB stem cells indicate overrepresentation of SG signature genes and divergence in uHF signature genes (from Joost et al. [2016]) in Ctr versus CerS4^epi−/− OB stem cells. NES, normalized enrichment score.