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. 2015 Dec 7;211(5):1057–1075. doi: 10.1083/jcb.201404147

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© 2015 Kumar et al.

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Figure 5. — Distinct epidermal barrier defects of KtyI^−/− and KtyII^−/−_K8 mouse embryos. (A) Toluidine blue assay indicating moderately delayed acquisition of dye-impermeable barrier in KtyI^−/− and a severe delay in KtyII^−/−_K8 compared with WT embryos. Bars, 5 mm. (B and C) 85% of CEs from E18.5 embryos were disrupted in KtyI^−/− compared with 99% in KtyII^−/−_K8 CE compared with WTs. Bars, 100 µm. (D) Immunofluorescence analysis of E18.5 epidermis revealing highly similar distribution of involucrin, loricrin, and filaggrin in KtyI^+/+ and KtyI^−/−. Note reduced filaggrin staining in KtyI^−/− samples. The basement membrane is indicated by white dotted lines. Bars, 20 µm. (E) Western blotting of skin extracts from E18.5 embryos showing a decrease in involucrin (Ivl) and absence of loricrin (Lor) and filaggrin (Flg) in KtyII^−/−_K8, together with decreased processing of profilaggrin (Pro-flg, upper bands) into filaggrin (bottom bands) in KtyI^−/− skin; α-tubulin (tub) served as loading control. (F) Transcriptome analysis exposing distinct changes in the expression of barrier protein-encoding genes in KtyI^−/− and KtyII^−/−. Note strong downregulation of a few barrier protein-encoding genes in KtyII^−/−_K8 only (in red); n = 3. (G) Quantitative RT-PCR analysis confirming the absence of transcription of loricrin, filaggrin, and hornerin genes in KtyII^−/−_K8 and upregulation of involucrin and loricrin mRNA in KtyI^−/− skin. ***, P ≤ 0.001.