PMC

(A) Graph of percentage wound closure relative to initial wound size in p73+/+ and p73-/- mice at 0, 3, 7, and 10 days after wounding. The mean area of eight wounds is shown with error bars representing SEM. (B) Dot plot of p73 H-score in unwounded and wounded (days 3, 7, and 10) skin specimens from p73+/+ mice. (C) Representative micrographs of IF staining for K14 (green), p73 (red), and DAPI (blue) in unwounded and post-wound day 7 skin specimens from p73+/+ mice. (D) Dot plot of the percentage of Ki67-positive cells in unwounded and wounded (days 3, 7, and 10) skin specimens from p73+/+ and p73-/- mice. (E) Representative micrographs of IF staining for Ki67 (red) and DAPI (blue) in skin specimens from p73+/+ and p73-/- mice 3 days after wounding. (F) Dot plot of γH2AX H-score in unwounded and wounded (days 3, 7, and 10) skin specimens from p73+/+ and p73-/- mice. (G) Representative micrographs of immunohistochemistry (IHC) staining for γH2AX in skin specimens from p73+/+ and p73-/- mice 10 days after wounding. All scale bars represent 50 μm. In (B), (D), and (F), horizontal lines represent the mean. In (C), (E), and (G), regions of the skin are labeled as: IFE, HF, epidermal wound edge (WE), and newly-formed epidermis of the wound (W); and the dotted line indicates the border between the WE and W. *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001. See also and Figs.

(A) Scatter plot of TP63 versus TP73 RNA-seq expression [units = transcripts per million (TPM)] by human tissue type (n = 37) from the Human Protein Atlas (172 total samples) []. Mean expression (TPM + 0.1) for each tissue is plotted on a log2 scale with a LOESS smooth local regression line (gray). Correlation between TP63 and TP73 was quantified using Spearman’s rank correlation coefficient (r_s). (B) Representative micrographs of H&E and immunofluorescence (IF) staining on serial human (top) and mouse (bottom) skin sections; DAPI (blue), p63α (green), and p73 (red). Regions of the skin in micrographs are labeled as: interfollicular epidermis (IFE), hair follicle (HF), outer root sheath (ORS), HF bulge (Bu), hair bulb (HB), sebaceous gland (SG), hair shaft (HS), and arrector pili muscle (APM). Scale bars represent 200 μm for human and 50 μm for murine tissue. See also and Figs.

(A) Immunoblot of KLF4, p63α, and p73 protein expression in neonatal human dermal fibroblast (HDFn) cells infected with lentivirus encoding ΔNp73 isoforms (ΔNp73α and ΔNp73β) or empty vector control in combination with KLF4 and ΔNp63α. Cells were grown for 3 days and protein was harvested for immunoblot analysis. (B) Bar graphs of RNA expression for the indicated keratinocyte genes in HDFn cells infected in (A). Cells were grown for 3 days and RNA was harvested for qRT-PCR analysis. Expression data are represented as the fold increase relative to control. The mean of three replicates is shown with error bars representing SEM. *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001. (C) Principal component analysis (PCA) plot of RNA-seq from HDFn cells infected with lentivirus encoding ΔNp73β or empty vector control in combination with KLF4 and ΔNp63α. Cells were grown for 6 days and RNA was harvested for RNA-seq analysis. The percentage of variance contributed by each PC is listed in parentheses. (D and E) Tables listing the enriched Genome Ontology (GO) categories and pathways among the top 250 genes contributing to PC1 from (C). (F) Heatmap with the expression of a set of 44 genes that underlie the enrichment of GO categories from (D). Genes are annotated based on known roles in iKC-related processes (gray box) and the presence of a p63/p73 ChIP-seq peak within 50 kb of its TSS in multiple basal cell types (brown box). See also and Figs and – Tables.

(A) UMAP (Uniform Manifold Approximation and Projection) plot of 2310 murine keratinocyte (back skin) single cell transcriptomes from the Tabula Muris dataset []. Each dot represents an individual cell and is colored according to cluster membership. (B) Bar graphs of RNA expression for keratinocyte cell type markers from (A). Each bar represents one cell and is colored by cluster number from (A). (C) Table summarizing Trp73 and Trp63 expression and co-expression in single murine keratinocyte cells from (A) by cluster number. Each cluster number is annotated with: the primary cell type of the cluster (based on cell type marker expression), the number (n) of cells belonging to the cluster, and the percentage of cells with each p73/p63 expression status. (D) Bar graph of the percentage of p73-positive cells in different classes of murine HF cells by single-cell RNA-seq (scRNA-seq) []. Hair cycle stages include: telogen, anagen I (Ana-I), anagen II (Ana-II), and anagen VI (Ana-VI). Cell types include: bulge HF stem cells (HFSC), hair germ (HG) cells, transit-amplifying cells (TAC), and dermal papilla (DP) cells. The number of cells analyzed for each cell type is listed above each bar. (E) Dot plot of Trp73 expression (TPM) in murine bulge HFSC and epidermal stem cells (SC) by bulk RNA-seq []. Cells were isolated by fluorescence-activated cell sorting (FACS) with marker-based sorting. Horizontal lines represent the mean. (F) Dot plot of TP73 expression (TPM) in primary human keratinocytes (HK) grown and differentiated in vitro for 6 or 7 days [,]. HKs were enriched for progenitors based on rapid collagen IV adherence. Horizontal lines represent the mean. (G) Table with the mean TP73 isoform expression in human skin (lower leg) RNA-seq samples (n = 473) from the Genotype-Tissue Expression (GTEx) Project [,]. For each sample, total gene abundance (TPM), N-terminal promoter usage, and C-terminal alternative splicing was calculated and is shown. See also .