Figure 6.
αII-spectrin–actomyosin networks regulate the EGFR–TRPV3–TGM pathway. (a) Dorsal skin sections from shCtr, and shSptan1 0595-transduced E17.5 embryos immunolabeled for pEGFR. Upper insets show transduced cells (H2B−GFP+). (b) Quantification of pEGFR granular layer intensity from data shown in a. Mean ± SD of 30 region of interest (ROI) from n = 3 embryos per condition. Horizontal bars: mean normalized intensity, dots: individual microscopy fields. **P = 0.0011 with Kolmogorov–Smirnov. (c) Quantification of ectopic pEGFR-positive cells from data shown in a. Horizontal bars: mean ± SD from n = 3 embryos per condition, dots: average ectopic pEGFR-positive cells per embryo. P = 0.1 with Kolmogorov–Smirnov. (d) Sections of dorsal skin from shCtr; TRPV3-GFP and shSptan1 0595; TRPV3-GFP–transduced E17.5 embryos. (e) Quantification of TRPV3-GFP cortical enrichment from the data shown in d. Mean ± SD from 30 individual cells from n = 3 embryos per condition. Bars: TRPV3-GFP cortex/cytoplasm ratio, dots: individual cells. ****P < 0.0001 with Kolmogorov–Smirnov. (f) ShCtr and shSptan1-0595–transduced primary mouse keratinocytes (H2B-GFP+) cultured in high-calcium (1.5 mM) medium and immunolabelled for E-cadherin and TRPV3. Insets (right) show magnifications of the boxed areas. (g) Quantification of E-cadherin and TRPV3 intensity. Mean ± SD from ∼200 mature junctions from n = 3 experiment per condition. Bars: mean normalized intensity, dots: mature junctions. *P < 0.05, ****P < 0.0001 with Kolmogorov–Smirnov. (h) Dorsal skin sections from E17.5 wild-type embryos treated with DMSO, latrunculin, or Y27632 immunolabeled for p-EGFR. (i) Quantification of pEGFR granular layer intensity from data shown in h. Mean ± SD of 30 ROI from n = 3 embryos per condition. Bars: mean normalized intensity; dots: individual microscopy fields with Kolmogorov–Smirnov. (j) Quantification of ectopic pEGFR-positive cells from data shown in h. Bars: mean ± SD from n = 3 embryos per condition. Dots: average ectopic pEGFR-positive cells from each embryo with Mann–Whitney. (k) Dorsal skin section from shCtr; TRPV3-GFP–transduced E17.5 embryos treated with DMSO, latrunculin or Y27632. Insets show the magnification of the boxed area from each epidermal layer. (l) Quantification of TRPV3-GFP cortical enrichment from the data shown in k. Data are the mean ± SD from 30 individual cells from n = 3 embryos per condition. Horizontal bars represent the TRPV3-GFP cortex/cytoplasm intensity ratio mean, and circles represent individual cells. ****P < 0.0001 with Kolmogorov–Smirnov. (m) Primary mouse keratinocytes cultured in high-calcium (1.5 mM) medium treated with DMSO, latrunculin, or Y27632 and immunolabelled for E-cadherin and TRPV3. (n) Quantification of TRPV3 intensity from data shown in c. Mean ± SD from ∼150 mature junctions from n = 3 experiment per condition. Bars: mean normalized intensity; dots: mature junctions. ****P < 0.0001 with Kolmogorov–Smirnov. (o) Dorsal skin sections from E17.5 wild-type embryos treated with DMSO, latrunculin, or Y27632 and processed for TGM activity assay. (p) Quantification of crosslinked TGM substrate intensity. Mean ± SD of 30 ROIs from n = 3 embryos per condition. Bars: mean normalized intensity; dots: individual microscopy fields. **P = 0.0072 (latrunculin B), *P = 0.0354 (Y27632) with Kolmogorov–Smirnov. (q) Quantification of cortically enriched cross-linked TGM substrate. Mean ± SD from 60 individual cells from n = 3 embryos per condition. Bars: means of cortex/cytoplasm ratio; dots: individual cells. ****P < 0.0001 with Kolmogorov–Smirnov. Nuclei were stained with DAPI; dotted lines indicate the dermal-epidermal border.

αII-spectrin–actomyosin networks regulate the EGFR–TRPV3–TGM pathway. (a) Dorsal skin sections from shCtr, and shSptan1 0595-transduced E17.5 embryos immunolabeled for pEGFR. Upper insets show transduced cells (H2B−GFP+). (b) Quantification of pEGFR granular layer intensity from data shown in a. Mean ± SD of 30 region of interest (ROI) from n = 3 embryos per condition. Horizontal bars: mean normalized intensity, dots: individual microscopy fields. **P = 0.0011 with Kolmogorov–Smirnov. (c) Quantification of ectopic pEGFR-positive cells from data shown in a. Horizontal bars: mean ± SD from n = 3 embryos per condition, dots: average ectopic pEGFR-positive cells per embryo. P = 0.1 with Kolmogorov–Smirnov. (d) Sections of dorsal skin from shCtr; TRPV3-GFP and shSptan1 0595; TRPV3-GFP–transduced E17.5 embryos. (e) Quantification of TRPV3-GFP cortical enrichment from the data shown in d. Mean ± SD from 30 individual cells from n = 3 embryos per condition. Bars: TRPV3-GFP cortex/cytoplasm ratio, dots: individual cells. ****P < 0.0001 with Kolmogorov–Smirnov. (f) ShCtr and shSptan1-0595–transduced primary mouse keratinocytes (H2B-GFP+) cultured in high-calcium (1.5 mM) medium and immunolabelled for E-cadherin and TRPV3. Insets (right) show magnifications of the boxed areas. (g) Quantification of E-cadherin and TRPV3 intensity. Mean ± SD from ∼200 mature junctions from n = 3 experiment per condition. Bars: mean normalized intensity, dots: mature junctions. *P < 0.05, ****P < 0.0001 with Kolmogorov–Smirnov. (h) Dorsal skin sections from E17.5 wild-type embryos treated with DMSO, latrunculin, or Y27632 immunolabeled for p-EGFR. (i) Quantification of pEGFR granular layer intensity from data shown in h. Mean ± SD of 30 ROI from n = 3 embryos per condition. Bars: mean normalized intensity; dots: individual microscopy fields with Kolmogorov–Smirnov. (j) Quantification of ectopic pEGFR-positive cells from data shown in h. Bars: mean ± SD from n = 3 embryos per condition. Dots: average ectopic pEGFR-positive cells from each embryo with Mann–Whitney. (k) Dorsal skin section from shCtr; TRPV3-GFP–transduced E17.5 embryos treated with DMSO, latrunculin or Y27632. Insets show the magnification of the boxed area from each epidermal layer. (l) Quantification of TRPV3-GFP cortical enrichment from the data shown in k. Data are the mean ± SD from 30 individual cells from n = 3 embryos per condition. Horizontal bars represent the TRPV3-GFP cortex/cytoplasm intensity ratio mean, and circles represent individual cells. ****P < 0.0001 with Kolmogorov–Smirnov. (m) Primary mouse keratinocytes cultured in high-calcium (1.5 mM) medium treated with DMSO, latrunculin, or Y27632 and immunolabelled for E-cadherin and TRPV3. (n) Quantification of TRPV3 intensity from data shown in c. Mean ± SD from ∼150 mature junctions from n = 3 experiment per condition. Bars: mean normalized intensity; dots: mature junctions. ****P < 0.0001 with Kolmogorov–Smirnov. (o) Dorsal skin sections from E17.5 wild-type embryos treated with DMSO, latrunculin, or Y27632 and processed for TGM activity assay. (p) Quantification of crosslinked TGM substrate intensity. Mean ± SD of 30 ROIs from n = 3 embryos per condition. Bars: mean normalized intensity; dots: individual microscopy fields. **P = 0.0072 (latrunculin B), *P = 0.0354 (Y27632) with Kolmogorov–Smirnov. (q) Quantification of cortically enriched cross-linked TGM substrate. Mean ± SD from 60 individual cells from n = 3 embryos per condition. Bars: means of cortex/cytoplasm ratio; dots: individual cells. ****P < 0.0001 with Kolmogorov–Smirnov. Nuclei were stained with DAPI; dotted lines indicate the dermal-epidermal border.

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