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Fig. 4 Human epidermal keratinocytes on different ECMs differentially regulate the expression of adherens junctions and desmosomes across layers in vitro. A Representative immunofluorescence images of P63, F-actin, and DAPI in both layers of the bilayer normal human epidermal keratinocytes (NHEK). DAPI image is z-depth color-coded. B Quantification of P63 intensity (two-tailed unpaired t-test, p < 0.0001, n = 15 (Basal) and 16 (Suprabasal) containing 5–20 cells/FOV (fields of view); 1 independent experiment). Box plot whiskers represent the minimum and maximum value. Bounds mark the 25th and 75th percentiles. Central line is the median. C Schematic of gel constructs containing 100% collagen (Collagen) and 100% collagen gel coated with laminin (Col (LM-coated)). D Representative immunofluorescence z-stack cross sections of NHEKs cultured on each gel system. E Representative immunofluorescence images of β-Catenin (CTNNB1) in culture on each gel type. Images of the Suprabasal, Interlayer (Suprabasal-Basal), and Basal shown. Intensity displayed using a 16-color scale. F Quantification of CTNNB1 intensity at junctions for Suprabasal, Interlayer, and Basal in NHEKs cultured on each gel system. For each graph, intensity was normalized to the Collagen Gel Group (two-tailed unpaired t-test (Suprabasal and Basal) and two-tailed Mann-Whitney test (Interlayer), p = 0.0046 (Suprabasal), 0.2213 (Interlayer), and p < 0.0001 (Basal), n= Suprabasal: 44 (Collagen) and 51 (Col LM coated) cells, Interlayer: 53 cells (Collagen and Col LM coated), and Basal: 41 (Collagen) and 44 (Col LM-coated) cells; 1 independent experiment). Data presented as box plots with whiskers to minimum and maximum value. Bounds mark the 25th and 75th percentiles. Central line is the median. G Representative immunofluorescence images of Desmoplakin (DSP) in culture on each gel type. Layers and intensity displayed as in (E). H Quantification of Desmoplakin intensity at junctions as in (F). (two-tailed unpaired t-test (Suprabasal and Interlayer) and two-tailed Mann-Whitney test (Basal), p = 0.0042 (Suprabasal) and p < 0.0001 (Interlayer and Basal), n= Suprabasal: 41 (Collagen) and 42 (Col LM coated) cells, Interlayer: 48 (Collagen) and 50 (Col LM coated) cells, and Basal: 40 (Collagen) and 42 (Col LM-coated) cells; 1 independent experiment). Data presented as box plots with whiskers to minimum and maximum value. Bounds mark the 25th and 75th percentiles. Central line is the median. Source data are provided as a Source Data file.