Figure S2.
E-cadherin controls cell shape upstream of spectrin. (a) Western blot analysis of primary mouse keratinocytes transduced with shCtr and Ank3 6780 shRNAs. Blots were probed for Ankyrin-3 and GAPDH. (b) qPCR analysis of Ank3 mRNA in primary mouse keratinocytes transduced with shCtr shRNA or Ank3-specific shRNAs (6780). Data are the mean ± SD of six preparations. **P = 0.002 with Kolmogorov–Smirnov. (c) Sagittal views of dorsal skin mosaic tissue sections from shAnk3 6780-transduced E17.5 embryos immunolabeled for Ankyrin-3. Graph: Quantification of Ankyrin-3 intensity. Mean ± SD of 60 individual cells from n = 3 embryos. Bars: mean normalized intensity; dots: individual cells. ****P > 0.0001 with Kolmogorov–Smirnov. (d) Sagittal views of dorsal skin sections from shCtr and shAnk3 6780-transduced E17.5 embryos immunolabeled for F-actin. Insets show transduced cells (H2B−GFP+). (e) Quantification of basal (left) and suprabasal (right) layer F-actin intensity from data shown in d. Data are the mean ± SD of 30 region of interest (ROI) from n = 3 embryos per condition. Horizontal bars represent the mean normalized intensity, and circles/squares represent microscopy fields. NS: P = 0.1344 (basal), NS: P = 0.9525 (suprabasal) with Kolmogorov–Smirnov. (f) Sagittal views of dorsal skin sections from shCtr and shAnk3 6780-transduced E17.5 embryos immunolabeled for E-cadherin. Insets show transduced cells (H2B−GFP+). (g) Quantification of basal (left) and suprabasal (right) layer cell cross-section area from data shown in f. Data are the mean ± SD from ∼200 individual cells from n = 3 embryos per condition. Horizontal bars represent the cell cross-section area mean, and circles/squares represent individual cells. NS: P = 0.2220 (basal), NS: P = 0.796 (suprabasal) by Kolmogorov–Smirnov. (h) Immunofluorescence analysis for the recruitment of E-cadherin and αII-spectrin to intercellular contacts in shCtr and shEcad 2287-transduced primary keratinocytes (GFP-positive nuclei, asterisks). Representative images of n = 3 biological replicates each. (i) Immunofluorescence analysis for E-cadherin (red) based on early intercellular contacts in shCtr and shSptan1 0595-transduced primary keratinocytes at the indicated Ca2+ time points. Representative images of n = 3 biological replicates each. (j) Phalloidin staining of single cells isolated from the granular layer treated with or without (nt) latrunculin B (0.1 µM). (k) Quantification of F-actin intensity (phalloidin) of isolated SG cells, as shown in j, treated with or without latrunculin B. Dots represent individual cells pooled from three mice. Source data are available for this figure: SourceData FS2.

E-cadherin controls cell shape upstream of spectrin. (a) Western blot analysis of primary mouse keratinocytes transduced with shCtr and Ank3 6780 shRNAs. Blots were probed for Ankyrin-3 and GAPDH. (b) qPCR analysis of Ank3 mRNA in primary mouse keratinocytes transduced with shCtr shRNA or Ank3-specific shRNAs (6780). Data are the mean ± SD of six preparations. **P = 0.002 with Kolmogorov–Smirnov. (c) Sagittal views of dorsal skin mosaic tissue sections from shAnk3 6780-transduced E17.5 embryos immunolabeled for Ankyrin-3. Graph: Quantification of Ankyrin-3 intensity. Mean ± SD of 60 individual cells from n = 3 embryos. Bars: mean normalized intensity; dots: individual cells. ****P > 0.0001 with Kolmogorov–Smirnov. (d) Sagittal views of dorsal skin sections from shCtr and shAnk3 6780-transduced E17.5 embryos immunolabeled for F-actin. Insets show transduced cells (H2B−GFP+). (e) Quantification of basal (left) and suprabasal (right) layer F-actin intensity from data shown in d. Data are the mean ± SD of 30 region of interest (ROI) from n = 3 embryos per condition. Horizontal bars represent the mean normalized intensity, and circles/squares represent microscopy fields. NS: P = 0.1344 (basal), NS: P = 0.9525 (suprabasal) with Kolmogorov–Smirnov. (f) Sagittal views of dorsal skin sections from shCtr and shAnk3 6780-transduced E17.5 embryos immunolabeled for E-cadherin. Insets show transduced cells (H2B−GFP+). (g) Quantification of basal (left) and suprabasal (right) layer cell cross-section area from data shown in f. Data are the mean ± SD from ∼200 individual cells from n = 3 embryos per condition. Horizontal bars represent the cell cross-section area mean, and circles/squares represent individual cells. NS: P = 0.2220 (basal), NS: P = 0.796 (suprabasal) by Kolmogorov–Smirnov. (h) Immunofluorescence analysis for the recruitment of E-cadherin and αII-spectrin to intercellular contacts in shCtr and shEcad 2287-transduced primary keratinocytes (GFP-positive nuclei, asterisks). Representative images of n = 3 biological replicates each. (i) Immunofluorescence analysis for E-cadherin (red) based on early intercellular contacts in shCtr and shSptan1 0595-transduced primary keratinocytes at the indicated Ca2+ time points. Representative images of n = 3 biological replicates each. (j) Phalloidin staining of single cells isolated from the granular layer treated with or without (nt) latrunculin B (0.1 µM). (k) Quantification of F-actin intensity (phalloidin) of isolated SG cells, as shown in j, treated with or without latrunculin B. Dots represent individual cells pooled from three mice. Source data are available for this figure: SourceData FS2.

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