Figure S1.
αII-spectrin determines epidermal cell shape. (a) From left to right: 3D whole mount of confetti epidermis from newborn mice showing expression of all four transgenic confetti colors, imaging of the cytoplasmic YFP signal, overlay of the rendering based on the YFP signal, and rendered cell volumes. (b) Quantification of cell sphericity from rendered cells per layer corresponding to Fig. 1 c. Dots: mean values per mouse. (c) Newborn epidermal whole-mount immunofluorescence analysis for phalloidin (F-actin), αII-spectrin, and tight junction marker occludin marking the SG2 layer. Overview of protein distribution across the layer corresponding to Fig. 1 c. Max. projections of the epidermal layers and a full projection (3D) are shown. (d) Immunofluorescence analysis for phalloidin (F-actin) and αII-spectrin on newborn epidermis cryosections of embryonic time points as indicated. Dashed line marks the epidermal-dermal boarder. (c and d) Representative images of N ≥ 3 biological replicates. (e) Quantitative qPCR analysis of Sptan1 mRNA in primary mouse keratinocytes transduced with Ctr shRNA or one of two Sptan1-specific shRNAs (0595 and 9753). Mean ± SD of six preparations. ****P < 0.0001 by unpaired t test. (f) Western blot analysis of primary mouse keratinocytes transduced with Scr, Sptan1 0595, or Sptan1 9753 shRNAs and quantification of αII-spectrin protein levels. Data are the mean ± SD of three preparations. ****P > 0.0001, ***P = 0.0006 by unpaired t test. (g) Dorsal skin mosaic tissue sections from shSptan1 0595-transduced E17.5 embryos immunolabeled for αII-spectrin. Line indicates areas of infected cells; dashed line indicates the dermal-epidermal border. Nuclei were stained with DAPI. Quantification of αII-spectrin intensity. Data are the mean ± SD of 60 individual cells from n = 3 embryos. Bars: mean normalized intensity; dots: individual cells. ****P > 0.0001 by unpaired t test. (h) Newborn epidermal whole-mount immunofluorescence analysis of αII-spectrin in Ctr and αII-spectrin–deficient epidermis (Sptan1epi−/−). Max. projection of the SG3 layer. (i) Western blot analysis of primary mouse keratinocytes isolated from Ctr and αII-spectrin–deficient epidermis (Sptan1epi−/−). (j) Immunofluorescence analysis for shape using combined staining for desmoglein1,2,3 (Dsg1,2,3) on Ctr and αII-spectrin–deficient newborn epidermis sections. (k) Quantification of cell sagittal area and shape (perimeter/√area)/layer using stainings as shown in j. ****P < 0.0001, **P < 0.005, and *P < 0.05; cells: n = 701 (basal), n = 927 (spinous), 688 (granular) with Kolmogorov–Smirnov per layer. Source data are available for this figure: SourceData FS1.

αII-spectrin determines epidermal cell shape. (a) From left to right: 3D whole mount of confetti epidermis from newborn mice showing expression of all four transgenic confetti colors, imaging of the cytoplasmic YFP signal, overlay of the rendering based on the YFP signal, and rendered cell volumes. (b) Quantification of cell sphericity from rendered cells per layer corresponding to Fig. 1 c. Dots: mean values per mouse. (c) Newborn epidermal whole-mount immunofluorescence analysis for phalloidin (F-actin), αII-spectrin, and tight junction marker occludin marking the SG2 layer. Overview of protein distribution across the layer corresponding to Fig. 1 c. Max. projections of the epidermal layers and a full projection (3D) are shown. (d) Immunofluorescence analysis for phalloidin (F-actin) and αII-spectrin on newborn epidermis cryosections of embryonic time points as indicated. Dashed line marks the epidermal-dermal boarder. (c and d) Representative images of N ≥ 3 biological replicates. (e) Quantitative qPCR analysis of Sptan1 mRNA in primary mouse keratinocytes transduced with Ctr shRNA or one of two Sptan1-specific shRNAs (0595 and 9753). Mean ± SD of six preparations. ****P < 0.0001 by unpaired t test. (f) Western blot analysis of primary mouse keratinocytes transduced with Scr, Sptan1 0595, or Sptan1 9753 shRNAs and quantification of αII-spectrin protein levels. Data are the mean ± SD of three preparations. ****P > 0.0001, ***P = 0.0006 by unpaired t test. (g) Dorsal skin mosaic tissue sections from shSptan1 0595-transduced E17.5 embryos immunolabeled for αII-spectrin. Line indicates areas of infected cells; dashed line indicates the dermal-epidermal border. Nuclei were stained with DAPI. Quantification of αII-spectrin intensity. Data are the mean ± SD of 60 individual cells from n = 3 embryos. Bars: mean normalized intensity; dots: individual cells. ****P > 0.0001 by unpaired t test. (h) Newborn epidermal whole-mount immunofluorescence analysis of αII-spectrin in Ctr and αII-spectrin–deficient epidermis (Sptan1epi−/−). Max. projection of the SG3 layer. (i) Western blot analysis of primary mouse keratinocytes isolated from Ctr and αII-spectrin–deficient epidermis (Sptan1epi−/−). (j) Immunofluorescence analysis for shape using combined staining for desmoglein1,2,3 (Dsg1,2,3) on Ctr and αII-spectrin–deficient newborn epidermis sections. (k) Quantification of cell sagittal area and shape (perimeter/√area)/layer using stainings as shown in j. ****P < 0.0001, **P < 0.005, and *P < 0.05; cells: n = 701 (basal), n = 927 (spinous), 688 (granular) with Kolmogorov–Smirnov per layer. Source data are available for this figure: SourceData FS1.

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