Fig. 4

Ablation of Sox11 and Sox4 induces premature epidermal differentiation (a) mRNA transcript copy number of SOXC class genes from FACS-purified E13.5 epidermal and P4 basal epidermal cells normalized to the housekeeping gene Mrpl19. Data are the mean ± SD. n = 2 biological replicates. b Images of E16.5 embryos of indicated genotypes in X-gal exclusion assay. Independent biological replicates of each genotype pair n = 3 Sox4 cKO-control pairs, 5 Sox11 cKO-control pairs, and 4 dcKO-control pairs in independent experiments. c Immunofluorescence analysis of epidermal differentiation marker expression in WT and dcKO at E16.5 (representative images from n = 3 biological replicates). FLG (filaggrin) and LOR (loricrin) are late differentiation markers expressed in termi80 (WT), 63 (Sox4 cKO), 83 (Sox11 cKO), or 88 (dcKO).nally differentiated keratinocytes in the epidermal outer layer; KRT1 (keratin 1) is an early differentiation maker expressed in cells of the intermediate epidermal layers. d BrdU incorporation analysis of WT and dcKO embryos. Pregnant mice were administered with BrdU for 4 h prior to embryo isolation. Skin sections of embryos at E16.5 were immunostained with anti-BrdU antibody. Representative sections are shown (left panel) and BrdU + cells in 13 fields were quantified (right panel). Data are the mean ± SD. n = 2 independent experiments. p = 0.51. e mRNA transcript copy numbers of SOXC class genes expressed in primary keratinocytes, normalized to the housekeeping gene Mrpl19. Cells were isolated from neonatal skins of WT and Sox4/11/12-triple knockout littermates. Data are the mean ± SD. n = 2 triple KO and 6 wild-type (WT) biological replicates. f qRT-PCR analysis of epidermal differentiation gene expression in WT, Sox11 cKO, Sox4 cKO, or dcKO primary keratinocytes. Keratinocytes were cultured in regular media or with the addition of calcium at 1.5 mM for 24 h. Data are the mean ± SD. n = 5 biological replicates. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant (Student’s one-tailed t-test). Scale bars, 5 mm (a), 50 µm (c, d). Source data for panels a and d–f are provided as a Source Data file