Cortical F-actin and spectrin organization are mutually dependent. (a) Sagittal views of dorsal skin sections from E17.5 wild-type embryos treated with DMSO, latrunculin, and Y27632 immunolabeled for E-cadherin. Nuclei were stained with DAPI; dotted lines indicate the dermal-epidermal border. NS, not significant. (b) Immunofluorescence analysis for αII-spectrin and F-actin after 6 or 48 h in high Ca²⁺ at cell–cell interfaces and apical surface. Single channels corresponding to Fig. 3 g. (c) Western blot analysis and quantification for αII-spectrin protein levels after Ca²⁺ switch for the time point indicated. Normalized to GAPDH and to 0 h Ca²⁺ time point. Dots represent biological replicates from n = 4 independent primary keratinocyte isolates. **P = 0.003 with Kruskal–Wallis, Dunn’s multiple comparison test. (d) Immunofluorescence analysis for E-cadherin, αII-spectrin, and F-actin after 48 h in high Ca²⁺ at cell–cell interfaces. (e) Immunofluorescence analysis for αII-spectrin and F-actin after 48 h in high Ca²⁺ at cell–cell interfaces with and without latrunculin B treatment (1 h, 0.1 µM). Single channels corresponding to Fig. 3 i. (f) Western blot analysis for αII-spectrin protein levels upon siRNA (siPOOLs)-mediated knockdown 96 h after transfection (72 h Ca²⁺). (g) Western blot quantification for αII-spectrin as shown in f, normalized to GAPDH. Dots represent biological replicates, n = 3 with Mann–Whitney. Representative example of n = 6 independent primary keratinocyte isolates. (h) Immunofluorescence analysis of F-actin organization at apical junction rings after 48 h in high Ca²⁺ and siRNA-mediated knockdown of αII-spectrin. (i) Quantification of F-actin intensity at apical junction rings (mean gray value, junctions/cytoplasm) as shown in h. Dots represent pooled values of single cells from n = 3 biological replicates. ****P < 0.0001 with Kolmogorov–Smirnov. Right graph: Mean values from n = 3 biological replicates tested with Mann–Whitney. Source data are available for this figure: SourceData FS3.