Fig 1: SFRP1 restrains keratinocyte progenitor proliferation.A Immunoblot of SFRP1 protein depletion in control (shControl) and SFRP1-depleted (shSFRP1) keratinocytes. Normalized SFRP1 relative expression is denoted. sgControl was set to 1 for comparation. B Colony morphology of shControl and shSFRP1 keratinocytes at endpoint of colony-formation assay. Scale bar, 1000 µm. c Crystal violet staining of colonies from shControl, shSFRP1, and shSFRP1 with recombinant human SFRP1 protein (rhSFRP1). Recombinant SFRP1 protein was applied to concentration of 1 ng/ml. D Quantification of colony formation area in (C). The average of shControl replicates was set to 1. Data are means ± SD (n = 3, one-way ANOVA with a Tukey’s honestly significant differences [HSD] post hoc test). p values are labeled above individual comparisons. E Quantification of colony formation number in (C). The average of shControl replicates was set to 1. Data are means ± SD (n = 3, one-way ANOVA with a Tukey’s honestly significant differences [HSD] post hoc test). F Ki67 immunofluorescence of control and SFRP1 knockdown keratinocyte colonies. Ki-67+ is labeled in green, and nuclei stained in blue. Scale bar, 300 µm. G Quantification of Ki-67+ cells in (F). Each datapoint represents the relative integrated density of Ki-67 signal. The median of shControl was set 1 for comparison. Data are median with interquartile range. (n = 862 and 1378 for shControl and shSFRP1, respectively, two-tailed unpaired student’s t-test). H UMAP visualization of all cell types or states identified in the human neonatal skin epidermis scRNA-seq dataset (n = 5). BAS basal keratinocytes, SPN spinous keratinocytes, GRN granular keratinocytes, APD skin appendage-related cells, MEL melanocytes, EC endothelial cells, LC Langerhans cells. I UMAP visualization of scRNA-seq data from human neonatal skin keratinocytes, colored by different cell states. These keratinocytes represent a subset derived from the neonatal foreskin epidermis UMAP in (H). Both non-proliferating (green dashed line) and proliferating (purple dashed line) basal keratinocytes are highlighted on the UMAP. BAS basal keratinocytes, SPN spinous keratinocytes, GRN granular keratinocytes. J Top: scaled and normalized expression of SFRP1, dividing cell marker, and skin basal keratinocyte marker genes; Bottom: feature plots for canonical markers of skin keratinocytes. K UMAP projection colored by cell-cycle phase. L Violin plot depicting the expression distribution of genes across different basal cell states. Horizontal bars within the violin plots indicate median values. M Scatter plot showing the co-expression analysis of MKI67 and SFRP1. Each point represents a single cell.
Fig 2: LIF promotes epidermal progenitor renewal and is inhibited by SFRP1.A Quantitative RT-PCR of SFRP1 and LIF mRNA in control vs. SFRP1-depleted keratinocytes across an in vitro differentiation time course (day 0-2-4). SFRP1_201, isoform SFRP1_201; SFRP1, total SFRP1; LIF_201, isoform LIF_201; LIF, total isoforms. Data are mean ± SEM. (n = 4, multiple paired student’s t-test). p values denoted above comparisons. B Immunoblot of LIF protein in control vs SFRP1-depleted primary keratinocytes across an in vitro differentiation time course. Relative intensity of LIF is denoted, using shSFRP1 at day 4 = 1.0 for comparison. C ELISA of LIF in control vs. SFRP1-depleted primary keratinocytes. shControl was set to 1 for comparison. Data are mean ± SEM. (n = 2, two-tailed ratio student’s t-test). D Immunoblot of SFRP1 in primary keratinocytes following control, SFRP1 knockdown (shSFRP1), and/or LIF knockdown (shLIF). Relative intensity of SFRP1 is denoted and normalized to beta tubulin. sgControl was set to 1 for comparison. E ELISA of LIF in primary keratinocytes following control, shSFRP1, and/or shLIF. Data are mean ± SD. shControl was set to 1 for comparison. (n = 3, one-way ANOVA with a Tukey’s HSD post hoc test). F Crystal violet staining of keratinocyte colonies generated following treatment with shControl, shSFRP1, shLIF, and/or recombinant human LIF (rhLIF). G Quantification of colony formation in (F). ShControl was set to 1 for comparison. Data are means ± SD. (n = 6, one-way ANOVA with a Tukey’s HSD post hoc test). H Immunoblot of primary keratinocyte protein lysates following treatment with shControl, shSFRP1, and/or ß-catenin knockdown (shCTNNB1). Relative intensity of SFRP1 and CTNNB1 is denoted and normalized to beta tubulin. shControl was set to 1 for comparison. I ELISA of LIF in primary keratinocytes following control, shSFRP1, and/or shCTNNB1. Data are mean ± SD. shControl was set to 1 for comparison. (n = 2, one way ANOVA with Tukey’s HSD). J Crystal violet staining of keratinocyte colonies generated following treatment with shControl, shSFRP1, and/or shCTNNB1. K Quantification of colony formation in (J). ShControl was set to 1 for comparison. Data are means ± SD. (n = 6, one-way ANOVA with a Tukey’s HSD post hoc test).
Fig 3: SFRP1 inhibits Wnt signaling and other stemness regulators.A Heatmap of differential gene expression in SFRP1-depleted keratinocytes. Differentially expressed genes (DEGs) between SFRP1-depleted and control keratinocytes were identified on day 0 and day 2 timepoints. DEGs were defined by threshold of p value ≤ 0.05 and | log2 (fold change)| > 1. B Top enriched GO terms (ranked by p-value) related to Wnt signaling. C Heatmap of gene expression for representative genes in (B). D Top enriched GO terms (ranked by p value) related to stem cell regulation. E Heatmap of gene expression for representative genes in (D). Genes associated with Wnt signaling are labeled in red. F Distribution of SFRP1-associated differential ATAC-seq peaks across genomic regions. Differential peaks were defined by threshold p value ≤ 0.05 and |fold change| > 1.5. G Top enriched Wnt GO (ranked by p value) of differential ATAC-seq associated genes. H Venn diagram of DEGs in RNA-seq and differential peak-associated genes in ATAC-seq. I Chromatin accessibility plot at the LIF genomic locus (chr22:30, 240, 453- 30, 246, 759). Shadowed fold change peaks meet threshold of >1.5 (shSFRP1 vs. shControl). Tracks were aligned with Ensembl LIF regulatory build and H3K27ac ChIP-seq data from foreskin keratinocytes.
Fig 4: SFRP1 knockdown in human epidermal organoids.A Schematic diagram of a working model of SFRP1 in the interfollicular epidermis. B Hematoxylin and eosin (H&E) staining on control and SFRP1-depleted epidermal organotypic tissues. Dotted black lines denote the basement membrane. Black arrowheads highlight example regions of collections of progenitor keratinocytes. Scale bar, 150 µm. C Quantification of basal curvature in (B). Each datapoint represents the curvature measurement of the basal layer of a sampled H&E image. Data are median ± interquartile range. (n = 30 from three independent biological replicates, two-tailed unpaired student’s t-test). D Quantification of epidermal area in (B). Each datapoint represents the relative area measurement of the epidermis of a sampled H&E image. Data are median ± interquartile range. (n = 30 from three independent biological replicates, two-tailed unpaired student’s t-test). E Immunofluorescence of epidermal organotypic tissues. Ki-67 marks proliferating keratinocytes. White dotted lines demarcate the basement membrane. Scale bar, 150 µm. F Quantification of Ki-67 positive cells in (E). Each datapoint represents the count of Ki-67+ cells from a sampled image, normalized to tissue length. Data are median ± interquartile range. (n = 30 from three biological replicates, evaluated with two-tailed unpaired student’s t-test). G Immunofluorescence of keratin 10, filaggrin, and loricrin in control and SFRP1-depleted epidermal organotypic tissues. Dotted white lines denote the basement membrane. Scale bar, 150 µm. H Quantitative RT-PCR of SFRP1 (isoform SFRP1_201, and total SFRP1) and differentiation-associated genes (KRT1, KRT10, FLG, LOR) in organoid epidermis in control and SFRP1 depleted tissues. I Immunoblot of FOXM1 in shControl and shSFRP1 primary keratinocytes over time course of in vitro differentiation. For relative quantitation, FOXM1 signal was normalized to beta tubulin, and the shControl day 0 signal was set to 1. J SFRP1 mRNA expression in skin diseases. Gene expression data from studies of keratinocyte cancers, psoriasis, and eczema were compiled. Selected studies included patient-matched normal skin (NS). Two-sided t-tests were performed to compare expression in NS vs. disease states. Data are mean ± SEM. Sample sizes (n) for each group depicted in figure. NS normal skin, AK actinic keratosis, IEC intraepidermal carcinoma, SCC squamous cell cancer, Pso psoriasis. P values shown above pairwise comparisons.
Fig 5: Silencing of SFRP1 prolongs anagen in healthy human HFs. Healthy human HFs were treated with 10 µM NTC siRNA, DKK1 siRNA, or SFRP1 siRNA for 5–8 days ex vivo. (a) Quantification of hair shaft production on days 1, 4, 5, 6, and 8 in culture with n = 24 (D1, D4), n = 6 (D5), n = 12–13 (D6), and n = 6 (D8) HFs from 1–4 independent donors. (b) Representative images of Ki-67/TUNEL immunofluorescence and Masson–Fontana histochemistry. (c) Quantitative analysis of hair cycle staging, n = 4 donors. (d) Quantitative analysis of the hair cycle score. Arbitrary units (au) were assigned to anagen (100), early catagen (200), mid catagen (300), and dystrophic (500) HFs. n = 23–24 HFs from 4 independent donors. (e,f) Quantitative analysis of hair matrix keratinocyte proliferation (e) and apoptosis (f). n = 23–24 HFs from 4 independent donors. All data are presented as mean ± SEM. Mann–Whitney test or unpaired t-test, * p < 0.05. Dots of different shapes indicate different donors. Data points reflecting the female donor are indicated by circles. DKK1: dickkopf 1, DP: dermal papilla, gHM: germinative hair matrix, HF: hair follicle, pcHM: pre-cortical hair matrix, SFRP1: secreted frizzled receptor 1.
Supplier Page from Abcam for Human SFRP1 ELISA Kit