Par3 loss in KCs increases P-cadherin surface levels and stability and enforces P-cadherin localization to heterologous KC–MC contacts. (A) P-cadherin immunostaining in Par3^KO KC–MC co-culture and MC monoculture. Bar, 10 µm. (B) P-cadherin immunostaining in control and Par3^KO co-cultures. Bar, 10 µm. (C) P-cadherin and Par3 coimmunostainings of neonatal control skin cross sections depicting interfollicular epidermis (top) and hair follicle (bottom) areas. Dashed lines demarcate the MC-containing basal epidermal layer. Bar, 10 µm. (D) P-cadherin and TRP2 immunohistochemistry of neonatal skin cross sections. Dashed lines underline the MC-containing basal epidermal layer. Bar, 10 µm. (A–D) Data are representative of three independent experiments. (E) Quantification of the number of P-cadherin–positive cells in interfollicular epidermis (IFE; data are mean ± SD; n = 3 mice per group; ****, P < 0.0001; Student’s t test) and MC number colocalizing with P-cadherin–positive cell clusters in hair follicles (data are pooled and depicted as mean ± SD; n = 5 mice per group; **, P = 0.0095; unequal variances Student’s t test with Welch’s correction). (F) Quantitative RT-PCR for P-cadherin RNA expression in control and Par3^KO primary KCs cultured at LC or 5 d HC. Data are pooled and represented as mean ± SEM. n = 5 biological replicates per group. LC: ns, P = 0.0940. HC: ns, P = 0.941. One-way ANOVA with Tukey posthoc test was used. (G) Western blot analysis for E- and P-cadherin in total lysates of control MCs and KCs, demonstrating that both cell types express both types of classical cadherins. Data are representative of three independent experiments. (H) Quantification of G. Cadherin levels were first normalized to GAPDH (loading control) before the expression in MCs relative to control KCs was determined. Data are pooled and represented as mean ± SD. n = 3 biological replicates per group. Student’s t test was used. (I) Biotinylation assay using primary KCs isolated from ctrl;HGF⁺;Cdk4^R24C/R24C and Par3^eKO;HGF⁺;Cdk4^R24C/R24C mice, 3 d after calcium switch, demonstrating elevated P-cadherin surface levels in Par3-deficient KCs. (J) Quantification of I. P-cadherin levels were normalized to GAPDH (loading control) before fold-changes relative to control cells were determined. Data are pooled and depicted as mean ± SEM. P- cadherin: n = 4 biological replicates per group; *, P = 0,0286; Mann Whitney U test. E- cadherin: n = 3 biological replicates per group. T: ns, P = 0.335; Surface: ns, P = 0.567; Student’s t test. (K) Immunoblot analysis of surface biotin-labeled P-cadherin stability in control and Par3^KO KCs cultured at 2 d HC. (L) Quantification of K. P-cadherin levels at 12-h chase period (after biotinylation) were normalized to total P-cadherin expression per genotype at 0 h (= pulse). Data are pooled and represented as mean ± SEM. n = 3 biological replicates per group. *, P = 0.0327; Student’s t test. Cad, cadherin; Ctrl, control; LE, long exposure; norm., normalized; PD, pull-down; pos., positive; rel., relative; T, total protein.