Fig 1: 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 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).
Supplier Page from Abcam for Human LIF ELISA Kit