Figure S5.
αII-spectrin regulates epidermal differentiation and barrier function. (a) Dorsal skin sections from shSptan1 0595-transduced E17.5 embryos immunolabeled for the basal layer marker K14 and the suprabasal marker K10. (b) Dorsal skin sections from shSptan1 9753-transduced E17.5 embryos immunolabeled for the basal layer marker K14 and the granular layer marker loricrin. Insets show the transduced cells (H2B−GFP+). (c) Dorsal skin sections from shSptan1 0595-transduced E17.5 embryos immunolabeled for the cleavage furrow marker survivin. Yellow lines show representative axes of division. Graph: Quantification of spindle orientation plotted as a cumulative frequency distribution. NS: P = 0.3485 with Mann–Whitney. (d) Dorsal skin sections from shSptan1 0595-transduced E17.5 embryos immunolabeled for EdU. (e) Quantification of EdU+ basal and suprabasal layer cells from the data shown in d. Bars: mean ± SD from n = 3 embryos per condition. Dots: average EdU+ basal and suprabasal layer cells from each embryo with Mann–Whitney. (f) Quantification of cell (nuclei) numbers from primary Ctr and Sptan1−/− or Sptan1 siRNA-treated keratinocytes differentiated for 48 h in high Ca2+. Dots: Mean values from biological replicates. >360 cells counted for Ctr/Sptan1−/− each and >20,000 cells for siCtr/siSptan1 each with Mann–Whitney. (g) Dorsal skin section from Ctr and Sptan1epi−/− newborn mice. Sections were processed for transglutaminase 1 (TGM1) activity assay or negative Ctr (mutated TGM substrate, pepQNK5). (h) Quantification of TGM1 intensity from data shown in g. Data are the mean of 30 fields of view from n = 3 newborn mice per condition. Bars: Mean intensity; dots individual microscopy fields. ****P < 0.0001 with Kolmogorov–Smirnov. (i) Dorsal skin sections from Ctr, Ecadepi−/− and Sptan1epi−/− newborn mice immunolabeled for total TGM1 protein. (j and k) Quantification of TGM1 intensity in Ctr, Ecadepi−/− and Sptan1epi−/−. Lines: Mean values/biological replicate. Nonsignificant with Mann–Whitney. (l) Dye exclusion assay: shCtr and shSptan1 9753-transduced E17.5 embryos were treated with toluidine blue dye to evaluate the skin barrier. (m) Dye exclusion assay: shCtr and shSptan1 0595-transduced E18.5 embryos were treated with toluidine blue.

αII-spectrin regulates epidermal differentiation and barrier function. (a) Dorsal skin sections from shSptan1 0595-transduced E17.5 embryos immunolabeled for the basal layer marker K14 and the suprabasal marker K10. (b) Dorsal skin sections from shSptan1 9753-transduced E17.5 embryos immunolabeled for the basal layer marker K14 and the granular layer marker loricrin. Insets show the transduced cells (H2B−GFP+). (c) Dorsal skin sections from shSptan1 0595-transduced E17.5 embryos immunolabeled for the cleavage furrow marker survivin. Yellow lines show representative axes of division. Graph: Quantification of spindle orientation plotted as a cumulative frequency distribution. NS: P = 0.3485 with Mann–Whitney. (d) Dorsal skin sections from shSptan1 0595-transduced E17.5 embryos immunolabeled for EdU. (e) Quantification of EdU+ basal and suprabasal layer cells from the data shown in d. Bars: mean ± SD from n = 3 embryos per condition. Dots: average EdU+ basal and suprabasal layer cells from each embryo with Mann–Whitney. (f) Quantification of cell (nuclei) numbers from primary Ctr and Sptan1−/− or Sptan1 siRNA-treated keratinocytes differentiated for 48 h in high Ca2+. Dots: Mean values from biological replicates. >360 cells counted for Ctr/Sptan1−/− each and >20,000 cells for siCtr/siSptan1 each with Mann–Whitney. (g) Dorsal skin section from Ctr and Sptan1epi−/− newborn mice. Sections were processed for transglutaminase 1 (TGM1) activity assay or negative Ctr (mutated TGM substrate, pepQNK5). (h) Quantification of TGM1 intensity from data shown in g. Data are the mean of 30 fields of view from n = 3 newborn mice per condition. Bars: Mean intensity; dots individual microscopy fields. ****P < 0.0001 with Kolmogorov–Smirnov. (i) Dorsal skin sections from Ctr, Ecadepi−/− and Sptan1epi−/− newborn mice immunolabeled for total TGM1 protein. (j and k) Quantification of TGM1 intensity in Ctr, Ecadepi−/− and Sptan1epi−/−. Lines: Mean values/biological replicate. Nonsignificant with Mann–Whitney. (l) Dye exclusion assay: shCtr and shSptan1 9753-transduced E17.5 embryos were treated with toluidine blue dye to evaluate the skin barrier. (m) Dye exclusion assay: shCtr and shSptan1 0595-transduced E18.5 embryos were treated with toluidine blue.

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