Figure 5.
αII-spectrin regulates epidermal differentiation and barrier function. (a) Dorsal skin from shCtr and shSptan1 0595-transduced E17.5 embryos immunolabeled for the basal layer marker K14, the differentiation marker K10, and the granular layer marker loricrin. Insets show the transduced cells (H2B−GFP+). (b) Quantification of cell area and shape of suprabasal shSptan1 K14+ and K14- cells in E17.5 embryos. Values of basal and suprabasal shCtr and basal shSptan1 are equal to Fig. 1 Lines: Mean values; dots: single cells pooled from 3 embryos with >100 cells for each condition. ****P < 0.0001 with Kruskal–Wallis, Dunn’s multiple comparison test. (c) Immunofluorescence analysis for the TJ marker occludin in Ctr and Sptan1−/− primary keratinocytes differentiated for 48 h in high Ca2+. Representative example of three biological replicates each. (d) Transepithelial resistance (TER) measurements in Ctr and αII-spectrin knockdown keratinocytes after switching to high Ca2+. Line represent means over time of three biological replicates each. Representative experiment of n > 10 biological replicates. (e) Newborn epidermal whole-mount immunofluorescence analysis for the TJ marker ZO-1, revealing impaired alignment of the upper old (red arrowheads) and the lower new TJ rings (blue arrowheads) in the granular layer 2 (SG2). Maximum projection of the granular layer. (f) Illustration of cell shapes and TJ organization in the SG2 of Ctr and Sptan1epi−/− epidermis. (g) Dorsal skin sections from shCtr and shSptan1 0595-transduced E17.5 embryos. Sections were processed for transglutaminase 1 (TGM1) activity assay. Upper Insets show the transduced cells (H2B−RFP+). (h) Quantification of TGM1 intensity from data shown in g. Data are the mean ± SD of 30 ROI from n = 3 embryos per condition. Bars: mean normalized intensity; dots individual microscopy fields. *P = 0.0471 by unpaired t-test. (i) Quantification of TGM1 activity cortical enrichment from the data shown in g. Mean ± SD from 60 individual cells from n = 3 embryos per condition. Bars: TGM1 activity cortex/cytoplasm intensity ratio mean; dots: individual cells. ***P = 0.0003 with Kolmogorov–Smirnov. (j) Dye exclusion assay: shCtr and shSptan1 0595-transduced E17.5 embryos were treated with toluidine blue dye to evaluate the skin barrier. (k) Dye exclusion assay: Ctr and Sptan1epi−/− E17.5 embryos were treated with toluidine blue dye to evaluate the skin barrier. (l) Dorsal skin section from Ctr and E-cadherinepi−/− newborn mice. Sections were processed for TGM1 activity assay or negative Ctr (mutated TGM substrate, pepQNK5). (m) Quantification of TGM1 intensity from data shown in l. (n) Transepidermal water loss (TEWL) measurements on Ctr and Sptan1epi−/− newborn mice. Dots represent individual mice. Data are the mean of 27 fields of view from n = 3 newborn mice per condition. Bars: Mean intensity; dots individual microscopy fields. ****P < 0.0001 with Kolmogorov–Smirnov. ROI, region of interest.

αII-spectrin regulates epidermal differentiation and barrier function. (a) Dorsal skin from shCtr and shSptan1 0595-transduced E17.5 embryos immunolabeled for the basal layer marker K14, the differentiation marker K10, and the granular layer marker loricrin. Insets show the transduced cells (H2B−GFP+). (b) Quantification of cell area and shape of suprabasal shSptan1 K14+ and K14- cells in E17.5 embryos. Values of basal and suprabasal shCtr and basal shSptan1 are equal to Fig. 1 Lines: Mean values; dots: single cells pooled from 3 embryos with >100 cells for each condition. ****P < 0.0001 with Kruskal–Wallis, Dunn’s multiple comparison test. (c) Immunofluorescence analysis for the TJ marker occludin in Ctr and Sptan1−/− primary keratinocytes differentiated for 48 h in high Ca2+. Representative example of three biological replicates each. (d) Transepithelial resistance (TER) measurements in Ctr and αII-spectrin knockdown keratinocytes after switching to high Ca2+. Line represent means over time of three biological replicates each. Representative experiment of n > 10 biological replicates. (e) Newborn epidermal whole-mount immunofluorescence analysis for the TJ marker ZO-1, revealing impaired alignment of the upper old (red arrowheads) and the lower new TJ rings (blue arrowheads) in the granular layer 2 (SG2). Maximum projection of the granular layer. (f) Illustration of cell shapes and TJ organization in the SG2 of Ctr and Sptan1epi−/− epidermis. (g) Dorsal skin sections from shCtr and shSptan1 0595-transduced E17.5 embryos. Sections were processed for transglutaminase 1 (TGM1) activity assay. Upper Insets show the transduced cells (H2B−RFP+). (h) Quantification of TGM1 intensity from data shown in g. Data are the mean ± SD of 30 ROI from n = 3 embryos per condition. Bars: mean normalized intensity; dots individual microscopy fields. *P = 0.0471 by unpaired t-test. (i) Quantification of TGM1 activity cortical enrichment from the data shown in g. Mean ± SD from 60 individual cells from n = 3 embryos per condition. Bars: TGM1 activity cortex/cytoplasm intensity ratio mean; dots: individual cells. ***P = 0.0003 with Kolmogorov–Smirnov. (j) Dye exclusion assay: shCtr and shSptan1 0595-transduced E17.5 embryos were treated with toluidine blue dye to evaluate the skin barrier. (k) Dye exclusion assay: Ctr and Sptan1epi−/− E17.5 embryos were treated with toluidine blue dye to evaluate the skin barrier. (l) Dorsal skin section from Ctr and E-cadherinepi−/− newborn mice. Sections were processed for TGM1 activity assay or negative Ctr (mutated TGM substrate, pepQNK5). (m) Quantification of TGM1 intensity from data shown in l. (n) Transepidermal water loss (TEWL) measurements on Ctr and Sptan1epi−/− newborn mice. Dots represent individual mice. Data are the mean of 27 fields of view from n = 3 newborn mice per condition. Bars: Mean intensity; dots individual microscopy fields. ****P < 0.0001 with Kolmogorov–Smirnov. ROI, region of interest.

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